Methods for wound healing

ABSTRACT

The invention relates to plasmids capable of expressing a protein targeting immune cells when transformed into a lactic acid bacterial cell, wherein the protein is chosen from the group consisting of murine and human CXCL12 1α; CXCL17 and Ym1. The invention further relates to lactic acid bacteria transformed with a said plasmid, as well as the use of said lactic acid bacteria for wound healing in humans and animals.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a National Phase patent application of InternationalPatent Application Number PCT/EP2015/081146, filed on Dec. 23, 2015,which claims priority of Swedish Patent Application 1451658-7, filedDec. 23, 2014. The entire contents of both of which are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy filed in the InternationalPatent Application number PCT/EP2015/081146, is named eolf-seql.txt andis 75,464 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to recombinant plasmids, and inparticular to plasmids capable of expressing a recombinant proteintargeting immune cells when transformed into a lactic acid bacterialcell, wherein the said protein is chosen from the group consisting ofmurine and human CXCL12 1α, CXCL17 and Ym1. The invention furtherrelates to lactic acid bacteria transformed with a said plasmid, as wellas the use of said lactic acid bacteria for wound healing in humans andanimals.

BACKGROUND TO THE INVENTION

The process of wound healing has overlapping phases (coagulation phase,inflammatory phase and proliferative/remodelling phase) whereconstituents of the local microenvironment change over time and distinctcell types play different roles. Key cell players in the healing processare platelets, keratinocytes/epithelial cells,fibroblasts/myofibroblasts, different immune cells and endothelialcells. All tissues in the body can be injured and the healing process issomewhat specific to the organ, however the initial signals elicited bythe damaged cells are similar. The most studied form of wound healing isin skin.

Tissue injury disrupts homeostasis, which initiates the coagulationprocess and activates the sympathetic nervous system. The plateletsforming the blood clots release signals, mainly PDGF (platelet derivedgrowth factor) and TGF (transforming growth factor) changing the localenvironment (Ref. 1). Injured and stressed cells release alarm signalsthat initiate the recruitment of immune cells such as neutrophils andmonocytes. Within the wounded tissue, the immune cells secrete variouschemokines, growth factors like VEGF-A, FGF, and EGF (vascularendothelial growth factor A, fibroblast growth factor, epidermal growthfactor), ROS (reactive oxygen species) and matrix digestive enzymes,which change the microenvironment and allow the healing process to enterthe proliferative phase where failing and dead tissue is removed bymacrophages. Cells from the wound edges, such as fibroblasts andkeratinocytes, migrate inwards to the wound centre and cover the woundsurface with a layer of collagen and extracellular matrix. Thefibroblasts within the wound are then transformed into myofibroblastsexpressing contractile α-SMA (α-smooth muscle actin) allowing the woundto contract and finally close. The transition from fibroblasts intomyofibroblasts is dependent on signals from the microenvironment, someof which originate from immune cells, mainly macrophages. During thisprocess, blood vessels are growing into the newly formed tissue, thegranulation tissue. Blood flow to the adjacent area is normallyincreased during this phase to increase the availability of oxygen andnutrients, in addition to immune cell recruitment and migration to theafflicted site.

Following wound closure, the afflicted site becomes re-epithelialized bykeratinocytes/epithelial cells whereby the integrity of the organbarrier is regained. Even after wound closure, some tissue remodellingoccurs to normalize the matrix structure and the majority of involvedimmune cells either die or leave the site. Also at this stage dead ordying cells are ingested and cleared (phagocytosed) by the remainingtissue macrophages (Ref. 1). Faster wound healing reduces complicationsand discomfort to the patient,

Impaired or delayed cutaneous or mucosal wound healing is a worldwideclinical problem causing pain, direct exposure to pathogens, loss oftissue function and loss of temperature and fluid balance regulation.There are several conditions where the tightly regulated wound healingprocess is impaired and the cutaneous or mucosal wounds remain unhealedfor longer time periods than normal, which in worst case become chronic.

Reduced blood flow to the skin, especially in extremities, significantlyreduces the efficiency of the healing process. There are severalclinical conditions where the skin perfusion is either reduced or thefunction of the vasculature is impaired such as PAD (peripheral arterydisease), intermittent claudication, vein insufficiency or vesselobstruction by arteriosclerotic plaques. Impaired blood flow to thewound area results in shortage of oxygen and nutrients and the cellsaiding in the tissue remodeling either die from necrosis or are unableto perform their tasks on site. Also the surrounding tissue will if notsufficiently supplied lose functionality and ultimately start to die.Tissues are during the remodeling phase very metabolically active andhave high oxygen consumption.

Another factor impairing cutaneous wound healing is hyperglycemia anddiabetes mellitus. During hyperglycemic conditions cell signaling andimmune system functions are impaired. Complications resulting fromdiabetes include microvascular changes and damage to peripheral neurons.As a result, diabetic patients often develop chronic wounds on theirfeet, commonly called diabetic foot. The available treatment for thesepatients today is removal of dead tissue using surgical debridement orcollagenase together with systemic antibiotic treatment and closed wounddressing. There are experimental studies where growth factors andbiomaterials have been applied to chronic wounds (Ref. 2).

The stromal cell-derived factor 1 (SDF-1) also known as C-X-C motifchemokine 12 (CXCL12) is a chemokine protein that in humans is encodedby the CXCL12 gene. WO 2009/079451 discloses a method for promotingwound healing in a subject, comprising administering directly to thewound or an area proximate the wound an amount of SDF-1 effective topromote healing of the wound of the subject.

Certain probiotics (Lactobacillus reuteri ATCC PTA 6475) have been shownto facilitate wound healing if supplemented in the drinking water duringthe healing process (Ref. 9), i.e. the bacteria were ingested. Further,supernatants from culture of Lactobacillus plantarum have beendemonstrated to inhibit biofilm production by Pseudomonas aeruginosa,commonly infecting chronic wounds (Ref. 10).

It has surprisingly been found that lactic acid bacteria which aremodified, according to the present invention, to express specificproteins, such as cytokines, are useful for promoting wound healing.Lactic acid bacteria are sparsely present on the human skin (Ref. 13)and are not the expected choice of bacteria to use for any interventionon the skin. Lactobacilli are difficult to work with since they growrelatively slowly and require special medium and conditions incomparison with more commonly used bacteria like E. coli and S. aureus.Further, Lactobacilli have limited intracellular machinery fortranscription, translation and protein folding. For this reason,nucleotide sequences coding for heterologous proteins have to beoptimized to fit the specific bacterial strain.

The different phases of wound healing comprise distinct key events thatcould be altered to change the healing process. Vascular remodelingduring the healing process is highly dependent on induction of hypoxiainducible factor 1α (HIF-1α) that regulates the expression of VEGF-A(vascular endothelial growth factor A) and a range of chemokines, suchas CXCL12 (also known as SDF-1; SEQ ID NO: 3 and 6). CXCL12 isconstitutively expressed in tissues and acts through the receptor CXCR4found on leukocytes and endothelial cells inducing multiple cellularactions (Ref. 3). CXCL12 is found in high levels in macrophagesspecialized in tissue remodeling (Ref. 4). Dermal overexpression ofCXCL12 using lentiviral vectors improves wound healing in diabetic mice(Ref. 5).

Another recently discovered chemokine is CXCL17 (SEQ ID NO: 9 and 12),which has similar effects on the phenotype of tissue macrophages asCXCL12. In similarity with CXCL12, CXCL17 is co-regulated with VEGF-Ameasured in cell culture (Ref. 6). CXCL17 is found mainly in mucosaltissues and have been reported to be directly antimicrobial topathogenic bacteria that are also found on skin whilst showing no effecton survival of Lactobacillus casei (Ref. 7).

A further protein of interest is Ym1 (SEQ ID NO: 15 and 18), which is achitinase-like protein. Chitin is a common polysaccharide in bacterialbiofilm. Ym1 both counteracts biofilm production and induces macrophagefunctions important for tissue remodeling and wound healing and isspecific to macrophages since it is not taken up by either vascularcells or epithelial cells (Ref. 8).

Consequently, in a first aspect the invention provides a recombinantplasmid which is capable of expressing a protein in lactic acid bacteria(i.e. when transformed into a lactic acid bacterial cell), wherein thesaid protein is useful for improving wound healing, such as cutaneous ormucosal wound healing, in a human or animal subject. Preferably, thesaid protein is useful for wound healing due to its capability oftargeting immune cells such as macrophages and their precursors.Preferably, the said protein is a cytokine or chemokine. Mostpreferably, the said protein is chosen from the group consisting ofmurine CXCL12, in particular murine CXCL12-1α (SEQ ID NO: 3); humanCXCL12, in particular human CXCL12-1α (SEQ ID NO: 6); murine CXCL17 (SEQID NO: 9); human CXCL17 (SEQ ID NO: 12); murine Ym1 (SEQ ID NO: 15); andhuman Ym1 (SEQ ID NO: 18).

This first aspect of the invention more particularly provides a plasmidwhich is capable of expressing a recombinant protein in lactic acidbacteria (i.e. when transformed into a lactic acid bacterial cell),wherein said plasmid comprises a nucleotide sequence encoding a proteinselected from CXCL12, CXCL17 and Ym1.

More specifically the nucleotide sequence may encode murine CXCL12, inparticular murine CXCL12-1α; human CXCL12, in particular humanCXCL12-1α; murine CXCL17; human CXCL17; murine Ym1; or human Ym1.

In one embodiment, the plasmid comprises a nucleotide sequence encodinga protein selected from murine CXCL12-1α having an amino acid sequenceas shown in SEQ ID NO: 3 or 2, or an amino acid sequence with at least80% sequence identity thereto; human CXCL12-1α having an amino acidsequence as shown in SEQ ID NO: 6 or 5, or an amino acid sequence withat least 80% sequence identity thereto; murine CXCL17 having an aminoacid sequence as shown in SEQ ID NO: 9 or 8, or an amino acid sequencewith at least 80% sequence identity thereto; human CXCL17 having anamino acid sequence as shown in SEQ ID NO: 12 or 11, or an amino acidsequence with at least 80% sequence identity thereto; murine Ym1 havingan amino acid sequence as shown in SEQ ID NO: 15 or 14, or an amino acidsequence with at least 80% sequence identity thereto; and human Ym1 asshown in SEQ ID NO: 18 or 17 or an amino acid sequence with at least 80%sequence identity thereto.

More particularly, the plasmid is for use in expressing a protein inlactic acid bacteria and is accordingly provided, or adapted, for suchuse (e.g. it is designed, selected, adapted or modified for specific orparticular use in lactic acid bacteria). Thus in one embodiment theplasmid is for specific expression in lactic acid bacteria, as comparedto bacteria or microorganisms generally. The plasmid may be adapted forexpression in lactic acid bacteria by means of its regulatory elements(regulatory sequences) and/or coding sequences, e.g. which are selectedor modified for expression in lactic acid bacteria.

Accordingly, in a more particular aspect the plasmid comprises one ormore regulatory (i.e. expression control) sequences which permitexpression, or which are specific for expression, in lactic acidbacteria. Thus, the plasmid may contain expression control sequencesderived from, or suitable for, or specific for, expression in lacticacid bacteria. Appropriate expression control sequences include forexample translational (e.g. start and stop codons, ribosomal bindingsites) and transcriptional control elements (e.g. promoter-operatorregions, termination stop sequences), linked in matching reading framewith the nucleotide sequence(s) which encode the protein(s) to beexpressed. The regulatory sequences(s) are operably linked to anucleotide sequence encoding said protein, such that they drive, orcontrol, expression of the protein. The plasmid may be introduced into alactic acid bacterial cell. Suitable transformation techniques are welldescribed in the literature. The bacterial cell may be cultured orotherwise maintained under conditions permitting expression of saidprotein from the plasmid. This may include conditions in a wound in asubject.

In one embodiment the promoter in the plasmid which controls expressionof the protein is a promoter which permits, or which is specific for,expression in lactic acid bacteria. Thus the plasmid may comprise anucleotide sequence(s) encoding the protein(s), under the control of (oroperably linked to) a promoter capable of expressing the protein inlactic acid bacteria. In a particular preferred embodiment the plasmidcomprises a lactic acid bacteria promoter, that is the promoter whichcontrols expression of the protein(s) is a promoter which is derivedfrom a lactic acid bacterium, or more particularly which is obtained orderived from a gene expressed in a lactic acid bacterium.

In some embodiments, in addition to a lactic acid bacterial promoter,the plasmid may also contain other regulatory elements or sequencesobtained or derived from lactic acid bacteria to control expression ofthe protein(s). Thus for example such other lactic acid bacterialexpression control elements or sequences may include enhancers,terminators and/or translational control elements or sequences asdiscussed above. In some embodiments the plasmid may also containregulatory elements or sequences which control or regulate expressionfrom the promoter e.g. operator sequences etc. or one or more regulatorygenes, as discussed further below.

Alternatively or additionally the plasmid may be adapted (or modifiedetc.) for use in lactic acid bacteria by virtue of the nucleotidesequences encoding the protein(s) being codon-optimised for expressionin lactic acid bacteria.

In a preferred embodiment the promoter for expression of the protein isa regulated (regulatable) or inducible promoter. Thus, expression of theprotein may be controlled or regulated (e.g. initiated, for example at adesired or appropriate time) by providing or contacting the bacteriawith a regulatory molecule or inducer which activates or turns on(induces) the promoter. This is advantageous in the context of deliveryof the protein to a wound.

Accordingly, a further aspect of the invention provides an expressionsystem for use in expressing a protein in lactic acid bacteria, saidexpression system comprising (i) a plasmid as defined herein, whereinsaid plasmid comprises a nucleotide sequence encoding a said proteinunder the control of an inducible promoter capable of expressing theprotein in lactic acid bacteria; and (ii) an inducer (or regulatorymolecule) for the promoter. The expression system may conveniently beprovided in the form of a kit comprising components (i) and (ii) above.

A still further aspect of the present invention is a bacterium, orbacteria, (i.e. a bacterial cell or strain) transformed with (i.e.comprising) a plasmid of the invention, as defined herein. Particularly,the bacterium is a lactic acid bacterium and the invention accordinglyprovides lactic acid bacteria (or a lactic acid bacterium) comprising aplasmid of the invention, as defined herein. Alternatively expressed,this aspect of the invention provides a bacterium (or bacterial cell)into which a plasmid of the invention has been introduced.

As described further herein, the plasmids and bacteria of the inventionare useful for promoting healing, and thus have particular utility inpromoting healing of wounds, which are defined herein to include injuredtissue generally (see further below). Accordingly further aspects of theinvention provide such plasmids and bacteria for use in therapy, andmore particularly for use in wound healing.

The bacteria may be provided for administration to a wound in a subjectto be treated in the form of a pharmaceutical composition. Accordingly astill further aspect of the invention provides a pharmaceuticalcomposition comprising bacteria of the invention as defined herein,together with at least one pharmaceutically acceptable carrier orexcipient.

More generally, the invention provides a probiotic product comprisingthe bacteria of the invention.

Such a product, or pharmaceutical composition, may conveniently take theform of a wound dressing comprising the bacteria of the invention. Thus,in a further aspect the invention provides a wound dressing comprisingbacteria of the invention as hereinbefore defined, together with atleast one dressing material.

A yet further aspect of the invention provides use of a plasmid or ofbacteria of the invention as defined herein for the manufacture of amedicament (or a probiotic product) for use in wound healing.

Also provided is a method of treating a subject to heal a wound, saidmethod comprising administering to said subject, or to the wound in saidsubject, an amount of bacteria of the invention as defined hereineffective to promote healing of the wound.

Another aspect of the invention provides a kit for healing wounds, saidkit comprising:

-   -   (i) lactic acid bacteria comprising a plasmid of the invention        as defined herein, wherein said plasmid comprises a nucleotide        sequence encoding a said protein under the control of an        inducible promoter capable of expressing the protein in lactic        acid bacteria; and    -   (ii) an inducer (or regulatory molecule) for the promoter.

A still further aspect of the invention comprises a pharmaceuticalproduct (e.g. a kit or combination product) comprising;

-   -   (i) lactic acid bacteria comprising a plasmid of the invention        as defined herein, wherein said plasmid comprises a nucleotide        sequence encoding a said protein under the control of an        inducible promoter capable of expressing the protein in lactic        acid bacteria; and    -   (ii) an inducer (or regulatory molecule) for the promoter,

as a combined preparation for separate, sequential or simultaneous usein wound healing (or for treating a wound in a subject).

The term “wound healing” is used broadly herein to include any aspect ofpromoting or improving the healing of a wound. Thus, the various aspectsof the invention set out above may alternatively be defined with respectto a utility of the plasmids or bacteria in promoting or enhancing orimproving wound healing or simply promoting or enhancing healing.

Wound healing may accordingly include or encompass any effect whichresults in faster wound healing, or more complete healing of a wound orindeed any amelioration or improvement in the healing of a wound, e.g.reduced healing time, for example reduced time to achieve partial orcomplete closure of a wound, improved wound appearance (e.g. theappearance of a healed or healing wound), reduced or improved scarformation, the promotion of healing of a chronic or recalcitrant woundetc. (i.e. the application of the bacteria of the invention to a woundmay induce, or cause, or start, the healing of a wound which has up tonow not healed or shown any signs of healing). Wounds are discussed inmore detail below.

The subject having a wound to be treated may be any human or animalsubject, including for example domestic animals, livestock animals,laboratory animals, sports animals or zoo animals. The animal ispreferably a mammalian animal, but other animals, e.g. birds areincluded. Thus the animal may be a primate, a rodent (e.g. a mouse orrat), or a horse, dog or cat. Most preferably the subject is a human.

Lactic acid bacteria (LAB) or Lactobacillales are a clade ofGram-positive, low-GC, acid-tolerant, generally nonsporulating,nonrespiring, either rod-shaped (bacillus), or spherical (coccus)bacteria which share common metabolic and physiological characteristics.These bacteria produce lactic acid as the major metabolic end product ofcarbohydrate fermentation and are characterized by an increasedtolerance to acidity (low pH range). These characteristics of LAB allowthem to outcompete other bacteria in a natural fermentation because LABcan withstand the increased acidity from organic acid production (e.g.lactic acid). Thus LAB play an important role in food fermentations, asacidification inhibits the growth of spoilage agents. Several LABstrains also produce proteinaceous bacteriocins which further inhibitspoilage and growth of pathogenic microorganisms. LAB have generallyrecognized as safe (GRAS) status and are amongst the most importantgroups of microorganisms used in the food industry.

The core genera that comprise the lactic acid bacteria clade areLactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus,as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus,Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, andWeissella. Any lactic acid bacteria from these genera are includedwithin the scope of the present invention, but particularly bacteriafrom the genera Lactobacillus or Lactococcus.

The plasmid may encode one or more of said proteins. Thus it may encodea combination of a CXCL12, CXCL17 and/or a Ym1 protein (e.g. 2 or moreof CXCL12, CXCL17 or Ym1). Alternatively, it may encode 2 or more typesof a CXCL12, CXCL17 and/or Ym1 protein (e.g. both murine and humanCXCL12 etc.). Where more than one protein is encoded, the protein may beexpressed from a nucleotide sequence encoding the proteins under thecontrol of a single promoter, or more than one promoter may be used. Forexample, each protein may be expressed from a separate promoter, whichmay be the same or different. Techniques for expression of 2 or moreproteins together from the same plasmid are well known in the art andinclude for example translational coupling techniques etc., means forachieving this are known and available in the art. For example multipletransgenes can be expressed simultaneously under one promoter using P2Aand T2A sequences.

The CXCL12, CXCL17 or Ym1 protein may be a native or natural protein(i.e. the nucleotide sequence may encode a protein having an amino acidsequence as found in nature) and may be from any species in which theseproteins occur. Generally the protein will be a mammalian protein and asindicated above human and murine proteins are preferred. However, thenative nucleotide sequences or protein sequences may be modified, forexample by one or more amino acid additions, insertions, deletionsand/or substitutions, as long as the function or activity of the proteinis not substantially or significantly altered, e.g. as long as theactivity of the protein is substantially retained. The protein may be afragment or truncated variant of a natural protein. For example, asequence-modified variant protein may exhibit at least 80, 85, 90 or 95%of the activity of the parent protein from which it is derived. This maybe assessed according to tests known in the art for activity of theprotein in question. For example, activity can be measured in systems ofreceptor phosphorylation or calcium flux upon ligation in culture cellstreated with the protein, in systems of cell chemotaxis in vitro or invivo in models of cell recruitment to the infected protein. An in vitroassay based on chemotaxis is described in Refs. 22 and 32. Ref. 33describes a further in vitro chemokine activity test which might beused. The terms “CXCL12”, “CXCL17” or “Ym1” thus include not only thenative proteins but also functionally equivalent variants or derivativesthereof. The proteins may thus be synthetic or sequence-modifiedvariants, or may comprise one or more other modifications, e.g.post-translational modifications etc.

As mentioned above, the encoded proteins may have the amino acidsequences indicated above for the native human or murine proteins,namely SEQ ID NOS. 3 and 6 for murine and human CXCL12 respectively, 9and 12 for murine and human CXCL17 respectively, and 15 and 18 formurine and human Ym1 respectively, or an amino acid sequence having atleast 80% sequence identity to any aforesaid sequence. Advantageously,as further indicated above, the nucleotide sequences encoding thesenative proteins may be codon-optimised for expression in lactic acidbacteria. This may result in a modified amino acid sequence of theprotein encoded. For example codon optimised sequences may encodesequences such as secretion sequences suitable, (or better suited) forlactic acid bacteria. Thus the “optimized” protein encoded by acodon-optimised nucleotide sequence may include an altered orsubstituted leader or signal sequence (e.g. secretory sequence) ascompared to the native protein. In a preferred embodiment the mature orcleaved form of the protein encoded by the codon optimised sequence isidentical to the native protein. Proteins encoded by codon-optimisednucleotide sequences may have an amino acid sequence as shown in SEQ IDNOS. 2, 5, 8, 11, 14, or 17 as listed in Table IV below. Thus, theprotein encoded by the plasmid may have an amino acid sequence as shownin SEQ ID NOS. 2 and 5 for murine and human CXCL12 respectively, 8 and11 for murine and human CXCL17 respectively, and 14 and 17 for murineand human Ym1 respectively, or an amino acid sequence having at least80% sequence identity to any aforesaid sequence.

In other embodiments the encoded protein(s) may have an amino acidsequence which has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%90%, 91% 92% 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity withany aforesaid amino acid sequence.

Sequence identity may readily be determined by methods and softwareknown and readily available in the art. Thus, sequence identity may beassessed by any convenient method. However, for determining the degreeof sequence identity between sequences, computer programs that makemultiple alignments of sequences are useful, for instance Clustal W(Ref. 24). Programs that compare and align pairs of sequences, likeALIGN (Ref. 25), FASTA (Ref. 26 and Ref. 27), BLAST and gapped BLAST(Ref. 28) are also useful for this purpose, and may be used usingdefault settings. Furthermore, the Dali server at the EuropeanBioinformatics institute offers structure-based alignments of proteinsequences (Ref. 29, Ref. 30 and Ref. 31). Multiple sequence alignmentsand percent identity calculations may be determined using the standardBLAST parameters, (e.g. using sequences from all organisms available,matrix Blosum 62, gap costs: existence 11, extension 1). Alternatively,the following program and parameters may be used: Program: Align Plus 4,version 4.10 (Sci Ed Central Clone Manager Professional Suite). DNAcomparison: Global comparison, Standard Linear Scoring matrix, Mismatchpenalty=2, Open gap penalty=4, Extend gap penalty=1. Amino acidcomparison: Global comparison, BLOSUM 62 Scoring matrix.

Variants of the naturally occurring polypeptide sequences as definedherein can be generated synthetically e.g. by using standard molecularbiology techniques that are known in the art, for example standardmutagenesis techniques such as site-directed or random mutagenesis (e.g.using gene shuffling or error prone PCR).

Derivatives of the proteins as defined herein may also be encoded. Byderivative is meant a protein as described above or a variant thereof inwhich the amino acid is chemically modified e.g. by glycosylation andsuch like etc.

Where a protein comprises an amino acid substitution relative to thesequence of the native protein, the substitution may preferably be aconservative substitution. The term “a conservative amino acidsubstitution” refers to any amino acid substitution in which an aminoacid is replaced (substituted) with an amino acid having similarphysicochemical properties, i.e. an amino acid of the same class/group.For instance, small residues Glycine (G), Alanine (A) Serine (S) orThreonine (T); hydrophobic or aliphatic residues Leucine (L), Isoleucine(I); Valine (V) or Methionine (M); hydrophilic residues Asparagine (N)and Glutamine (Q); acidic residues Aspartic acid (D) and Glutamic acid(E); positively-charged (basic) residues Arginine (R), Lysine (K) orHistidine (H); or aromatic residues Phenylalanine (F), Tyrosine (Y) andTryptophan (W), may be substituted interchangeably without substantiallyaltering the function or activity of the protein.

As indicated above, it is preferred to use an inducible promoter forexpression of the protein. By “inducible” is meant any promoter whosefunction (i.e. activity, or effect in allowing or causing transcriptionof the coding nucleotide sequence) can be regulated or controlled. Theterm “inducible” is thus synonymous, and may be used interchangeablywith “regulatable” (or “regulated”). Thus, there is not constitutiveexpression of the protein. Accordingly, expression of the protein may beinduced, or turned on (or more particularly turned on and off). Moreparticularly, expression may be induced, or turned on for a finite ordefined time. This may be because expression ceases after a period oftime, and/or because the bacterial cells die.

In some embodiments there may be no expression (transcription) from thepromoter until the promoter is induced (or alternatively termed,activated). However, as with any biological system, lack of activity maynot be absolute and there may be some basal promoter activity in theabsence of promoter activation or induction. However, in a preferredembodiment any basal expression of the uninduced promoter is low,minimal, or insignificant, or more preferably de minimis or negligible.Thus, expression from the inducible promoter is advantageouslymeasurably or demonstrably increased when the promoter is inducedcompared to the promoter when it is not induced.

Inducible promoters are well known in the art, including induciblepromoters for use in lactic acid bacteria and any appropriate induciblepromoter may be used, suitable for expression in lactic acid bacteria.

An inducible promoter may be induced (or activated) in the presence ofan inducer or activator molecule, which may act directly or indirectlyon the promoter, and which may be added to induce the promoter, or moregenerally to cause or enable induction or activation of the promoter,and permit expression of the protein, or it may be induced (oractivated) by a change in conditions of the bacteria containing theplasmid, e.g. by introducing a change of conditions to the lactic acidbacteria, e.g. starvation or depletion of a particular nutrient. Aninducer of the promoter may be encoded by a regulatory gene, which in anembodiment may itself be induced or activated. The term “inducer” isthus used broadly herein to include any regulatory molecule, or indeedany permissive condition, which may activate or turn on an induciblepromoter, or allow or cause an inducible promoter to be induced. Thus,induction of an inducible promoter may comprise the introduction of(e.g. contacting the lactic acid bacteria containing the plasmid with) aregulatory molecule or of a condition permissive to promoter induction(activation). In some embodiments the inducer can be an activationpeptide. This may act directly, or indirectly to induce the promoter,for example as described further below.

As noted above, promoters obtained or derived from lactic acid bacteriaare preferred. These may be native promoters or modified or mutantpromoters. A suitable promoter may for example be identified by growinglactic acid bacteria in a wound, and by determining which genes areexpressed, or upregulated in the bacteria in the wound. The promotersfrom such genes may then be identified. Alternatively a number ofdifferent promoters and expression systems in or for use in lactic acidbacteria have been identified and described or available in the art,including expression plasmids containing such promoters or expressionsystems for use in LAB. Any such known plasmid or expression system maybe used as the basis for the recombinant plasmid of the invention.

Various inducible expression systems are known in the art for use withLAB such as Lactobacilli. One example includes an auto-inducing systembased on the manganese starvation-inducible promoter from the manganesetransporter of L. plantarum NC8 as described in Ref. 19. This systemdoes not require the addition of external inducers for recombinantprotein production.

Duong et al. (Ref. 20) describe expression vectors for use withlactobacilli based on the broad range pWV01 replicon and containingpromoters from operons involved in fructooligosaccharide (FOS), lactoseor trehalose metabolism or transport, or in glycolysis. Such promotersmay be induced by their specific carbohydrate and repressed by glucose.

More particularly, the inducible expression system may compriseinducible promoters involved in the production of LAB proteins, and inparticular bacteriocins. The activity of such promoters may becontrolled by a cognate regulatory system based on the bacteriocinregulon, for example a two-component regulatory (signal transduction)system which responds to an externally added activator peptide (i.e. aninducer/regulatory molecule in peptide form) and genes encoding ahistidine protein kinase and response regulator necessary to activatethis promoter upon induction by an activator peptide.

In an embodiment the expression system may be based on thenisin-controlled expression (NICE) system, based on the combination ofthe nisA promoter and the nisRK regulatory genes. This system is basedon the promoters and regulatory genes from the Lactococcus lactis nisingene cluster and has been used to develop regulated gene expressionsystems for lactococci, lactobacilli and other Gram-positive bacteria(reviewed briefly in Ref. 15 and Ref. 21). Whilst the NICE systems areefficient and well regulated in Lactococci, these systems can exhibitsignificant basal activity. This can be circumvented by integrating thehistidine kinase and response regulator genes in the chromosome,limiting the expression systems to specially designed host strains.

In another embodiment the expression system may be based on the genesand promoter involved in the production of class II bacteriocins sakacinA (sap genes) by the sakacin A regulon or sakacin P (spp genes) by thesakacin P regulon. Such vectors are known as pSIP vectors and contain apromoter element derived from either the sakacin A or the sakacin Pstructural gene with an engineered NcoI site for translational fusioncloning. A variety of such vectors containing different promoters fromthe regulons and/or different replicons are described in Ref. 21 andRef. 15 and any of these vectors could be used as the basis for therecombinant plasmid of the invention.

In a representative embodiment the promoter may be the P_(sapA),P_(sppA) or P_(orfX) promoter from the sakacin A or P regulon, togetherwith its associated or cognate regulatory genes.

In a particularly preferred embodiment the plasmid contains the pSH71replicon, the P_(orfx) promoter from the sakacin P regulon and thecognate regulatory genes, based on the vector pSIP411 depicted in FIG.12 and described in Ref. 21. Plasmid pSIP411 is designated pLAB112 inthe present application. The inducer for use in such an embodiment ispreferably an activation peptide based on the peptide SppIP, e.g. anactivation peptide having the sequence of SEQ ID NO: 19, or an aminoacid sequence with at least 80% (or more particularly at least 85, 90 or95) sequence identity thereto. In a preferred embodiment the saidrecombinant plasmid is derived from the plasmid designated pLAB112having the nucleotide sequence shown in SEQ ID NO: 20.

The use of an inducible promoter (or inducible expression system) mayprovide the advantage of a more controlled, and in particular prolongedexpression of the protein in the wound setting i.e. when the bacteriaare administered to the subject or to the wound. For a better effect inpromoting wound healing it is advantageous for the protein to beexpressed by the bacteria for a period of time at the site of the wound(e.g. at the wound surface), e.g. for at least 40, 45, 50, 55 or 60minutes, notably for at least one hour, or more. Thus the protein may beexpressed for a finite, a defined or a prolonged period of time. Resultspresented in the Examples below show that using plasmids and bacteriaaccording to the present invention, the protein may be expressed for aperiod of about an hour at the wound surface. The plasmids and bacteriamay in some embodiments be optimised to allow expression of the protein(e.g. in a wound) for 2, 3 or 4 hours or more.

Continuous expression and delivery of the protein is thus desirable andthis may be afforded by using the transformed bacteria of the invention.By “continuous” or “prolonged” is meant that there is expression, andhence delivery, of the protein over a period of time e.g. over a periodof at least an hour (or so, as discussed above). In particular thisallows the protein to be effective over a period of time which isincreased as compared to administration of the protein directly (i.e. asa protein product rather than by expression by the bacteria).

As discussed above, the nucleotide sequences encoding the protein(s) maybe codon optimised for expression in LAB. Accordingly, in preferredembodiments the nucleotide sequences (or inserts) in the recombinantplasmids, which encode the proteins, may be selected from thecodon-optimised nucleotide sequences shown in SEQ ID NOS. 1, 4, 7, 10,13 and 16 which encode murine CXCL12, human CXCL12, murine CXCL17, humanCXCL17, murine Ym1 and human Ym1 respectively, or a nucleotide sequencehaving at least 80% sequence identity therewith.

Thus in representative embodiments the recombinant plasmid may be chosenfrom the group consisting of the plasmids designated mLrCK1, comprisinga nucleotide sequence as shown in SEQ ID NO: 1; mLrCK1.4, comprising anucleotide sequence as shown in SEQ ID NO: 1; mLrCK1.7, comprising anucleotide sequence as shown in SEQ ID NO: 1; hLrCK1, comprising anucleotide sequence as shown in SEQ ID NO: 4; mLrCK2, comprising anucleotide sequence as shown in SEQ ID NO: 7; hLrCK2, comprising anucleotide sequence as shown in SEQ ID NO: 10; hLrMP1, comprising anucleotide sequence as shown in SEQ ID NO: 13; and mLrMP2, comprising anucleotide sequence as shown in SEQ ID NO: 16.

In some embodiments the plasmid of the invention comprises a nucleotidesequence which has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89% 90%, 91% 92% 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identityto a nucleotide sequence of the following codon optimized inserts mLrCK1(i.e., to the nucleotide sequence of SEQ ID NO: 1), mLrCK1.4 (i.e., tothe nucleotide sequence of SEQ ID NO: 1), mLrCK1.7 (i.e., to thenucleotide sequence of SEQ ID NO: 1), hLrCK1 (i.e., to the nucleotidesequence of SEQ ID NO: 4), mLrCK2 (i.e., to the nucleotide sequence ofSEQ ID NO: 7), hLrCK2 (i.e., to the nucleotide sequence of SEQ ID NO:10), hLrMP1 (i.e., to the nucleotide sequence of SEQ ID NO: 13), andmLrMP2 (i.e., to the nucleotide sequence of SEQ ID NO: 16).

Sequence identity of nucleotide molecules may be determined by methodsand software known and widely available in the art, for example by FASTASearch using GCG packages, with default values and a variable pamfactor,and gap creation penalty set at 12.0 and gap extension penalty set at4.0 with a window of 6 nucleotides.

Such sequence identity related nucleotide sequences may be functionallyequivalent to the nucleotide sequence which is set forth in SEQ ID NO:1, 4, 10, 13 or 16. Such nucleotide sequences may be consideredfunctionally equivalent if they encode polypeptides which would beconsidered functional equivalents to the respective CXCL12, CXCL17 orYm1 proteins. Preferred functional equivalents are those which encodethe preferred proteins as set out above.

In another aspect, the invention provides a bacterial strain transformedwith the recombinant plasmid described above. The said bacterial strainis preferably a lactic acid bacteria strain such as a Lactobacillusstrain or a Lactococcus (e.g. Lactococcus lactis) strain. Morepreferably, the bacterial strain is a Lactobacillus reuteri strain suchas Lactobacillus reuteri R2LC or Lactobacillus reuteri DSM20016. Thesaid strains (Lactobacillus reuteri R2LC/DSM20016 and Lactococcuslactis) are not found on human skin as determined by phylogeneticanalysis of the forearm skin biota of six subjects (Ref. 13).

As well as the plasmids, expression systems, bacteria and kits, furtherproducts of the invention include pharmaceutical compositions andmedical devices containing the bacteria. Such compositions and devicesmay include in particular wound dressings, packing materials, swabs,implants etc., or indeed any wholly or partially in-dwelling medicaldevice which may be introduced or present at the site of a wound (e.g.at a surgical wound site), for example a line or catheter or implant.Also included are probiotic products, that is products containing thebacteria for administration to a subject, e.g. for oral administration,for example for consumption or ingestion, or for topical application toa wound or direct administration to a wound site, e.g. during surgery,or rectally, vaginally, etc.

Accordingly, the products (e.g. plasmids, bacterial strains, probioticsand wound dressings etc.) according to the invention are useful inmedical therapy, in particular for promoting wound healing in human oranimal subjects. As used herein, the term “promoting wound healing”means augmenting, improving, increasing, or inducing closure, healing,or repair of a wound. In preferred aspects of the invention, the humanor animal subject is in need of wound healing due to an underlyingmedical condition leading to impaired wound healing, such as reducedperipheral blood perfusion (peripheral artery disease), hyperglycemia orneuropathy, or the subject may be immunocompromised for any reason, e.g.due to an underlying disease (whether acquired or inherited) or as aneffect of medical treatment. In particular the subject may be sufferingfrom diabetes.

The wound to be healed can include any injury, trauma or damage to anyportion of the body of a subject. Examples of wounds that can be treatedby the method include acute conditions or wounds; such as thermal burns(hot or cold), chemical burns, radiation burns, electrical burns, burnscaused by excess exposure to ultraviolet radiation (e.g. sunburn);damage to bodily tissues, such as the perineum as a result of labor andchildbirth; injuries sustained during medical procedures, such asepisiotomies, trauma-induced injuries including cuts, incisions,excoriations; injuries sustained from accidents; post-surgical injuries,as well as chronic conditions; such as pressure sores, bedsores, ulcers,conditions related to diabetes and poor circulation, and all types ofacne. In addition, the wound can include dermatitis, wounds followingdental surgery; periodontal disease; wounds following trauma; and tumorassociated wounds. Further examples are gastrointestinal woundsoccurring during for instance gastritis or inflammatory bowel disease.

Thus the term “wound” is used broadly herein to include any breach ofthe integrity of a tissue or any damage or injury to a tissue. Thus theterm includes any damage, trauma or injury to tissue or any lesion,howsoever caused (e.g. due to accidental injury or trauma, surgical orother intended or purposeful injury or disease). The trauma may includeany physical or mechanical injury or any damage caused by an externalagent including pathogens or biological or chemical agents. Tissuedamage may also be caused by hypoxia, ischemia or reperfusion. Woundsmay include any type of burn. The wound may be acute or chronic. Achronic wound may be described as any wound stalled in a healing stage,e.g. in the inflammatory phase, or any wound that has not healed in 30,40, 50 or 60 days or more. The wound may be present in or on an internalor external surface or tissue of the body.

In a particular embodiment the wound is on an external surface or tissueof the body, e.g. it is a skin (i.e. cutaneous) wound or a mucosalwound, in particular a wound in an external mucosal tissue or surface ofthe body (e.g. in the eye, ear or nose etc.) In another embodiment it isa gastrointestinal wound. In a different embodiment it is not agastrointestinal wound (i.e. it is a wound other than a gastrointestinalwound).

The bacteria may be administered in any convenient or desired way, e.g.orally, or topically, or by direct administration to a wound site e.g.by direct injection or infusion or application or introduction of apharmaceutical composition or dressing or device containing thebacteria. In other embodiments it may be administered to the oralcavity, or intranasally or by inhalation, rectally or vaginally. Thebacteria may thus be administered to, or via, any orifice of the body.For administration to a gastrointestinal wound the bacteria may beadministered perorally.

The bacteria may be formulated or prepared in any convenient or desiredway for administration by any of the above routes, according toprocedures and using means well known and routine in the art. Thus, aswell as pharmaceutical compositions, medical devices and dressings etc.,the probiotic products of the invention may be formulated and providedas or in nutritional supplements or foods, e.g. functional foods.

Oral administration forms include powders, tablets, capsules and liquidsetc. For topical administration, the product may be formulated as aliquid e.g. a suspension, or a spray or aerosol (powder or liquid), gel,cream, lotion, paste, ointment or salve etc. or as any form of dressing,e.g. bandage, plaster, pad, strip, swab, sponge, mat etc., with orwithout a solid support or substrate. Further the bacteria may beprovided on (e.g. coated on) the surface of a medical device such as animplant (e.g. a prosthetic implant), tube, line or catheter etc.

The bacteria may be provided in any convenient or desired form, e.g. asan active or growing culture or in lyophilized or freeze-dried form.

The bacterial strains according to the invention can be formulated fortopical or oral administration to treat surface wounds of skin ormucosa. Consequently, the invention further provides a probiotic productcomprising a bacterial strain according to the invention. The saidprobiotic product is e.g. a pharmaceutical composition, preferably fororal administration. Alternatively, for topical application, theprobiotic product is e.g. a lotion or a lotion-soaked wound dressing,comprising a bacterial strain according to the invention.

The product of the invention (i.e. the pharmaceutical composition ordevice or dressing etc.) may also contain the inducer (where aninducible promoter is used). This may be provided as part of the product(e.g. incorporated into or included in a dressing) or separately, e.g.as part of a kit or combination product, as defined above.

When co-formulated together in a product (e.g. a dressing or device) thebacteria and the inducer may be provided in a format in which thebacteria are separated from the inducer and are brought together (orcontacted) in use. For example, the bacteria and inducer may be inseparate compartments which are brought together (e.g. contacted ormixed), or may be separated by a barrier (e.g. a membrane or otherpartition) which may be broken or disrupted or opened in use.

Alternatively, the inducer may be formulated and provided separately(e.g. in a kit also containing the bacteria, or a product containing thebacteria), and the inducer and bacteria (or product containing thebacteria) may be brought together (e.g contacted) during use. This maybe before, during or after administration to the subject. For example, aproduct comprising the bacteria may be administered first and then theinducer may be added or applied to the bacteria. In another embodimentthe bacteria and inducer may be premixed before administration, e.g.just before or immediately before, or during administration.

Where bacteria are provided in lyophilized or freeze-dried form, it maybe desirable to reconstitute, or resuspend, them prior to administratione.g. prior to or during use. This may depend on the wound and the formatof the product which is used. For example, in the case of some woundsthere may be sufficient liquid present to allow for the bacteria to bereconstituted/resuspended and become active. However, in otherembodiments it may be desirable to provide a liquid for reconstitution(or alternatively expressed, for suspension or resuspension) of thebacteria. This may be provided in a separate vessel or container (e.g.as part of a kit or combination product) or in a separate compartment ofa container, or vessel or device. The liquid may comprise or contain theinducer, or the inducer, when present, may be provided in a separatevessel or container or compartment. The liquid may be any suitableliquid for reconstitution or suspension of freeze-dried bacteria, e.g.water, or an aqueous solution, or buffer or growth or culture medium.

Thus, by way of example a two compartment system (e.g. in a dressing ordevice or container or vessel (e.g. a bottle)) may comprise freeze-driedbacteria in one compartment and a liquid in another. The liquid mayoptionally contain an inducer. In use, or prior to use, the twocompartments may be mixed or brought into contact, and applied to thewound. In a more particular embodiment, the bacteria may be administeredto a wound in liquid form, and a separate dressing may then be applied.It will be seen therefore that in one simple embodiment, a kit maysimply contain a first vessel or container comprising the freeze-driedbacteria and a second vessel or container containing a liquid forreconstitution of the bacteria. Optionally the kit may also contain aninducer, which is also contained in the second vessel or in separatethird vessel or container.

Hence, for example a said probiotic product preferably comprises anactivation peptide capable of activating expression of the protein to beexpressed in the lactic acid bacteria strain. The said activationpeptide is preferably the peptide SppIP (i.e. a peptide comprising theamino acid sequence of SEQ ID NO: 19, or a sequence with at least 80%sequence identity thereto).

For cutaneous wounds, the said wound dressing can comprise freeze-driedbacteria in one compartment and an activation peptide in anothercompartment. When applied to the wound, the two compartments are broughttogether so that the bacteria are brought into contact with theactivation peptide. Alternatively, bacteria can be contained in agel-like structure on the adhesive side of a waterproof plaster or theside of the dressing in contact with the exudate. At the time of use,activation peptide is manually applied to the bacteria and the plasteror dressing is applied to the wound area.

Viable bacteria may also be comprised in a hydrocolloid, for example anatural gelatin. The bacteria can be incorporated by crosslinking intohydrocolloid e.g. gelatin films, plasticised and dried, retainingviability during storage until hydration. Viable bacteria may also beencapsulated within cross-linked electrospun hydrogel fibers. In thisformat the bacteria need not be freeze-dried.

For wounds in the gastrointestinal tract, a tablet is designedcomprising at least two separate compartments, wherein one compartmentcomprises freeze-dried bacteria and the other compartment comprisesliquid and an activation peptide. The tablet is squeezed beforeingestion so that an inner membrane, separating the two compartments, isbroken and the contents are mixed together. For wounds in the mouth(e.g. on the gums), bacteria according to the invention can beadministered in a high viscous paste.

Specifically, formulations for topical administration to the skin caninclude ointments, creams, gels, and pastes to be administered in apharmaceutically acceptable carrier. Topical formulations can beprepared using oleaginous or water-soluble ointment bases, as is wellknown to those in the art. For example, these formulations may includevegetable oils, animal fats, and more preferably semisolid hydrocarbonsobtained from petroleum. Particular components used may include whiteointment, yellow ointment, acetyl esters wax, oleic acid, olive oil,paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite,white wax, yellow wax, lanolin, anhydrous lanolin, and glycerylmonostearate. Various water-soluble ointment bases may also be usedincluding, for example, glycol ethers and derivatives, polyethyleneglycols, polyoxyl 40 stearate, and polysorbates.

The bacterial strain can be provided in and/or on a substrate, solidsupport, and/or wound dressing for delivery of active substances to thewound. The solid support or substrate may be a medical device or a partthereof. As used herein, the term “substrate” or “solid support” and“wound dressing” refer broadly to any substrate when prepared for, andapplied to, a wound for protection, absorbance, drainage, etc.

In an embodiment the invention provides a wound healing material ordressing attached to or comprising the transformed bacterial strain i.e.the dressing is a vehicle for administering the transformed bacteria ofthe invention. Alternatively the vehicle may be a plaster or bandage.The present invention may include any one of the numerous types ofsubstrates and/or backings that are commercially available, the choiceof wound healing material will depend on the nature of the wound to betreated. The most commonly used wound dressings are described brieflybelow.

Transparent film dressings are made of e.g. polyurethane, polyamide, orgelatin. These synthetic films are permeable to water vapor oxygen andother gases but impermeable to water and bacteria, have low absorbencyand are suitable for wounds with low exudate), hydrocolloids(hydrophilic colloidal particles bound to polyurethane foam), hydrogels(cross-linked polymers containing about at least 60% water have higherabsorbency and eliminate toxic components from the wound bed andmaintain the moisture level and temperature in the wound area), foams(hydrophilic or hydrophobic e.g. polymeric foam dressings producedthrough the modification of polyurethane foam have good absorbency andare permeable to water vapour), calcium alginates (non-woven compositesof fibers from calcium alginate from the phycocolloid group, alginateshave a very high absorbent capacity. They also promote autolyticdebridement because ion-exchange between the alginate and the exudateconverts the insoluble calcium alginate into soluble sodium alginate,providing the wound bed with a moist, intact surface ideal for woundhealing), and cellophane (cellulose with a plasticizer). The shape andsize of a wound may be determined and the wound dressing customized forthe exact site based on the measurements provided for the wound. Aswound sites can vary in terms of mechanical strength, thickness,sensitivity, etc., the substrate can be molded to specifically addressthe mechanical and/or other needs of the site. For example, thethickness of the substrate may be minimized for locations that arehighly innervated, e.g. the fingertips. Other wound sites, e.g. fingers,ankles, knees, elbows and the like, may be exposed to higher mechanicalstress and require multiple layers of the substrate.

In yet a further aspect, the invention provides a method for woundhealing in a human or animal subject, comprising administering to ahuman or animal subject in need thereof a bacterial strain according tothe invention. The said bacterial strain is preferably comprised in apharmaceutical composition or wound dressing as hereinbefore described.In such methods, the human or animal subject is preferably in need ofwound healing due to an underlying medical condition leading to impairedwound healing, such as reduced peripheral blood perfusion (peripheralartery disease), hyperglycemia or neuropathy.

Results obtained and included in the Examples below demonstrate theadvantages of the invention. In particular, improved wound healing (e.g.in terms of better or faster wound closure) may be obtained by using theprotein-expressing transformed bacteria of the invention, as comparedto, for example, a protein preparation directly (i.e. just the protein,no bacteria) or just bacteria alone (bacteria which are not modified toexpress the protein, e.g. not containing the recombinant plasmid).Further, an improved effect may be seen when bacteria are administeredto wound, compared to administration of a supernatant obtained from atransformed bacterial culture. It is thus advantageous to deliver theprotein to the wound by means of a lactic acid bacterial host expressingthe protein. It is believed that there may be synergistic effect. Inother words there may be a synergy between the effect of the bacteriaand the effect of the protein on wound healing. Accordingly, in someembodiments there may be a greater than cumulative effect of thetransformed bacteria on wound healing, relative to the effect ofcorresponding untransformed bacteria (i.e. not containing the plasmid)and the effect of the protein when provided as a protein (i.e. notexpressed from bacteria in situ).

It is believed in this respect that the effect of the bacteria inlowering pH e.g. in the site of the wound may assist in augmenting orenhancing or promoting the activity of the protein. Whilst not wishingto be bound by theory, it is further believed that administration of thetransformed bacteria according to the invention may have a beneficialeffect in promoting macrophage activity at the site of the wound. Forexample, the number of macrophages may be increased.

The effect of the transformed bacteria on wound healing may or may notbe immediate, and may take some time to be seen (e.g. 1, 2, 3, 4, 5 or 6or more hours to be seen, or longer, e.g. 8, 10, 12, 15, 18, 20 or 24hours or more, or 1, 2, 3, 4, 5 or 6 or more days to be seen, or longere.g. 8, 10, 12, 15, 18, 20 or 24 days or more, before improved woundhealing can be observed). For chronic wounds in elderly humans it maytake longer to see a difference between the treatment group and controlgroup for example it may take around 12 weeks.

A particular and important utility of the present invention lies in thetreatment of chronic wounds, particularly ulcers and in particular inthe treatment of diabetic foot ulcers.

The prevalence of chronic foot ulcers in persons with diabetes is about18%. In 2013, the European population reached 742.5 million, whichtranslates into 32.7 million with diabetes, of which 2.9-5.8 millionhave chronic foot ulcers. Mean duration of an ulcer of this type is inthe range of months where less than 25% of the wounds are healed within12 weeks when standard care is given. The end stage of this condition isamputation of the affected limb. Today the treatment of people havingchronic foot ulcers is divided between primary care, home care, nursinghomes, relatives, self-management and visits to hospital wound clinics.The current treatment relies on off-loading, removal of dead tissueusing surgical debridement, repeated changes of wound dressings,systemic antibiotics and in special cases treatment with living larvaeor collagenase and at a few locations in Sweden hyperbaric oxygentreatment can be offered. If an underlying cause also includesobstructions of larger arteries, this can be corrected surgically bybypassing vein graft. Today the wounds are treated every second to thirdday. Treatment with the suggested modified lactic acid bacteria in anyof the suggested forms or formulations would not disrupt this practicalroutine. Improved healing of such wounds by the treatments of thepresent invention would thus be of considerable economic benefit, aswell as of personal benefit to the patient.

The bacteria are active and produce and deliver the encoded proteins tothe wound surface for a period of time (e.g. about one hour) in vivo.They may then become inactive and die. Slow or dead lactic acid bacteriacan with no risk be in the wound/dressing environment until the dressingis changed as normal.

The biotherapeutic according to the present invention will havesignificantly lower production cost compared to protein drug compounds.This is because the biotherapeutics produces the active protein itselfdirectly in the wound.

Open wounds such as diabetic foot ulcers, together with loss of functionin the foot, cause considerable discomfort, and even disability to thepatient, and can have a significant negative impact on quality of life,including significant risk or infection and therefore prolonged use ofantibiotics, and ultimately amputation. Improved healing would thus beof tremendous personal benefit to the patient and would also have thebenefit of reducing antibiotic use (and consequently the spread ofantibiotic resistance). It is believed that treating such chronic woundsaccording to the invention may amplify endogenous alarm signals in thewound, and kick start the healing process in stalled or chronic wounds,and accelerate healing time.

Further, the invention may have advantages in flexibility and ease ofuse by medical staff.

BRIEF DESCRIPTION OF THE DRAWINGS

Representative methods and preferred embodiments according to thepresent invention will be further described with reference to thefollowing non-limiting Examples and Figures in which:

FIGS. 1A & 1B. Growth (FIG. 1A) and pH (FIG. 1B) over time in mLrCK1Lactococcus lactis re-inoculated from overnight culture at start OD0.285 and 0.51 with addition of 10 or 50 ng/ml promoter activationpeptide SppIP.

FIG. 2. Expression of pLAB112_Luc in Lactobacillus reuteri R2LCre-inoculated from overnight culture at start OD 0.5 in vitro measuredby bioimaging over time. A baseline image at time 0 was acquired.Promoter activation peptide SppIP (50 ng/ml) and substrate D-Luciferin(150 μg/ml) were added immediately after. The plate was imaged at 5minutes and then every 30 minutes for 1400 minutes. Media used in allsamples is MRS. Peptide is promoter activation peptide SppIP. Each groupconsists of eight samples.

FIG. 3. Expression of pLAB112_Luc in Lactobacillus reuteri R2LCre-inoculated from overnight culture to start OD 0.5 applied on 1 dayold cutaneous full thickness wounds. In vivo expression measured bynon-invasive bioimaging over time. A baseline image at time 0 wasacquired on 5 anesthetized mice with 1 day old cutaneous full thicknesswounds. Then 25 μl Lactobacillus reuteri R2LC_pLAB112_Luc activated withpromoter activation peptide SppIP (50 ng/ml) and substrate D-Luciferin(150 μg/ml) was added to the middle of the wounds and mice were imagedat 5 minutes and then every 15 minutes for 270 minutes.

FIGS. 4A, 4B & 4C. Time to wound healing in healthy mice. Time to 50%(FIG. 4A), 75% (FIG. 4B) or complete (100%) (FIG. 4C) healed woundsurface, n=5 all groups. FIGS. 4A, 4B, 4C, One-way ANOVA, Bonferronicompare all columns.

FIGS. 5A & 5B. Wound size (FIG. 5A) and wound exposure (FIG. 5B) overtime in healthy mice. Wound size measured daily from images with a scaleincluded, n=5 all groups. FIG. 5A, Two-way ANOVA, Bonferroni compare allcolumns, d0-d5 analyzed. Change due to time and treatment. Decreasedwound size by R2LC_pLAB112_LrCK1.4 at d1 and d2 compared to Controls.FIG. 5B, One-way ANOVA, Bonferroni compare all columns, all daysanalyzed. Decreased wound exposure by R2LC_pLAB112_LrCK1.4 for the wholehealing process.

FIGS. 6A & 6B. Ischemia induction by femoral artery ligation prior towound induction, n=4 in all groups. Cutaneous blood flow measured inischemic limb (FIG. 6A) and the contralatheral corresponding unaffectedlimb (FIG. 6B) of anesthetized mice over time using Laser SpeckleContrast Analysis. Data is expressed in perfusion unites (PFU). A and B,Two-way ANOVA, Bonferroni compare all columns, d0-d7 analyzed. No changeis observed due to time or treatment.

FIGS. 7A, 7B & 7C. Time to wound healing in mice with ischemia at thetime of wound induction. Time to 50% (FIG. 7A), 75% (FIG. 7B) orcomplete (100%) (FIG. 7C) healed wound surface, n=4 all groups. FIGS.7A, 7B & 7C, One-way ANOVA, Bonferroni compare all columns.

FIGS. 8A & 8B. Wound size and wound exposure over time in mice withlocal ischemia at the time and location of wound induction. Wound sizemeasured daily from images with a scale included, n=4 all groups. FIG.8A, Two-way ANOVA, Bonferroni compare all columns, d0-d7 analyzed.Change due to time and treatment. Decreased wound sizeR2LC_pLAB112_LrCK1.4 at d1 and d2 compared to Controls. FIG. 8B, One-wayANOVA, Bonferroni compare all columns, all days analyzed. Decreasedwound exposure by R2LC_pLAB112_LrCK1.4 for the whole healing process.

FIGS. 9A & 9B. Body weight (FIG. 9A) and blood glucose (FIG. 9B)following induction of diabetes using a single i.v. injection of alloxanmonohydrate. Diabetic Controls, n=4, Diabetic R2LC_pLAB112_Luc, n=5,Diabetic R2LC_pLAB_LrCK1.4, n=4. FIGS. 9A and 9B, Two-way ANOVA,Bonferroni compare all columns, d0-d6 analyzed. No change was observeddue to time or treatment.

FIGS. 10A, 10B & 10C. Time to wound healing in mice with diabetes atwound induction. Time to 50% (FIG. 10A), 75% (FIG. 10B) or complete(100%) (FIG. 10C) healed wound surface, Diabetic Controls, n=4, DiabeticR2LC_pLAB112_Luc, n=5, Diabetic R2LC_pLAB_LrCK1.4, n=4. FIGS. 10A, 10B &10C, One-way ANOVA, Bonferroni compare all columns.

FIGS. 11A & 11B. Wound size and wound exposure over time in mice withdiabetes at wound induction. Wound size measured daily from images witha scale included, Diabetic Controls, n=4, Diabetic R2LC_pLAB112_Luc,n=5, Diabetic R2LC_pLAB_LrCK1.4, n=4. FIG. 11A, Two-way ANOVA,Bonferroni compare all columns, d0-d6 analyzed. Change due to time. FIG.11B, One-way ANOVA, Bonferroni compare all columns, all days analyzed.No diff. (p=0.08).

FIG. 12. The pSIP411 plasmid.

FIG. 13. Quantification of plasmid expression in dermis in wound edge(40 μg DNA) using detection of luminescent signal by non-invasivebioimaging (IVIS Spectrum) over 11 days (n=10).

FIGS. 14A, 14B & 14C. Time to wound healing in healthy mice. Time to 50%(FIG. 14A), 75% (FIG. 14B) or complete (100%) (FIG. 14C) healed woundsurface (n=8 pCTR, n=9 pCXCL12). FIGS. 14A, 14B & 14C, Students unpairedtwo-tailed t-test.

FIGS. 15A & 15B. Wound size (FIG. 15A) and wound exposure (FIG. 15B)over time in healthy mice. Wound size measured daily from images with ascale included (n=8 pCTR, n=9 pCXCL12). (FIG. 15A) Two-way ANOVA,Bonferroni compare all columns, d0-d7 analyzed. Change due to time.(FIG. 15B) Students two-tailed unpaired t-test. Tendency (p=0.08) todecreased wound exposure by pCXCL12 for the whole healing process.

FIG. 16. Measurements of bacterial concentrations for Lactobacillusreuteri R2LC expressed as optical density (OD) and colony forming unitsper ml (CFU/ml).

FIGS. 17A & 17B. Wound size (FIG. 17A) and wound exposure (FIG. 17B)over time in healthy mice treated with different concentrations ofLactobacillus reuteri R2LC_pLAB112_LrCK1.4. Wound size measured dailyfrom images with a scale included. FIG. 17A, Two-way ANOVA, Bonferronicompare all columns, d0-d2 analyzed. Change due to time and treatment.(FIG. 17A) Two way ANOVA Bonferroni compare all columns, (FIG. 17B) OneWay ANOVA Bonferroni compare all columns (p<0.05). Decreased woundexposure by treatment with Lactobacillus reuteri R2LC_pLAB112_LrCK1.4 atOD 0.2, 0.5, 1.0 and 1.25 as compared to wound receiving no treatment.(Control, n=15; OD 0.2, n=4; 0.5, n=10, OD 1.0, n=4; OD 1.25, n=5).

FIGS. 18A & 18B. Wound size (FIG. 18A) and wound exposure (FIG. 18B)over time in healthy mice treated with different concentrations ofmurine CXCL12 1α at one time point per day for two days. Wound sizemeasured daily from images with a scale included. FIG. 18A, Two-wayANOVA, Bonferroni compare all columns, d0-d2 analyzed. Change due totime. FIG. 18B, wound exposure the first two days. (Control, n=15; 0.2μg CXCL12 1α, n=4; 0.6 μg CXCL12 1α, n=5, 1.0 μg CXCL12 1α, n=4).

FIGS. 19A & 19B. Wound size (FIG. 19A) and wound exposure (FIG. 19B)over time in healthy mice treated with 0.2 μg recombinant protein every10^(th) minute for one hour every day. Wound size measured daily fromimages with a scale included. FIG. 19A, Two-way ANOVA, Bonferronicompare all columns, d0-d2 analyzed. Change due to time. FIG. 19B, woundexposure the first two days. (No treatment, n=15; CXCL12 1α, n=6;CXCL17, n=9, Ym1, n=9).

FIGS. 20A & 20B. Re-epithelialization measured in human skin epidermalpunch biopsy wounds. FIG. 20A shows pH measured in culture medium after24 hours of culturing skin discs with epidermal wounds with no treatmentor treatment with LB_Luc or LB_LrCK1. FIG. 20B shows length of the newlyformed epidermis sleeve growing from the wound edge over the exposeddermis after 14 days of culture. * indicates difference, One Way ANOVABonferroni compare selected columns (p<0.05).

FIG. 21. In vitro expression of pLAB112_Luc in Lactobacillus reuteriR2LC immediately after revival from freeze-dried state measured in vitroby bioimaging over time. A baseline image at time 0 was acquired. Thenpromoter activation peptide SppIP (50 ng/ml) and substrate D-Luciferin(150 μg/ml) was added immediately after. The plate was imaged at 5minutes and then every 5-15 minutes for 930 minutes. Media used in allsamples is MRS. Peptide is promoter activation peptide SppIP. Each groupconsists of four samples.

FIG. 22. In vivo expression of pLAB112_Luc in Lactobacillus reuteri R2LCimmediately after revival from freeze-dried state and application on 1day old cutaneous full thickness wounds measured in vivo by bioimagingover time. A baseline image at time 0 was acquired on three anesthetizedmice with two separate 1 day old cutaneous full thickness wounds. Then25 μl Lactobacillus reuteri R2LC_(—) pLAB112_Luc activated with promoteractivation peptide SppIP (50 ng/ml) and substrate D-Luciferin (150μg/ml) was added to the middle of the wounds and mice were imaged at 5minutes and then every 15 minutes for 270 minutes.

FIGS. 23A & 23B. Wound size (FIG. 23A) and wound exposure (FIG. 23B)over time in healthy mice treated with freeze-dried, revived and inducedLactobacillus reuteri R2LC_pLAB112_LrCK1.4. Wound size measured dailyfrom images with a scale included. (FIG. 23A) Two-way ANOVA, Bonferronicompare all columns, d0-d2 analyzed. Change due to time and treatment.(FIG. 23B) One Way ANOVA Bonferroni compare all columns (p<0.05).Decreased wound size was observed following treatment with Lactobacillusreuteri R2LC_pLAB112_LrCK1.4 compared to Lactobacillus reuteriR2LC_pLAB112_Luc also when the bacteria had been freeze-dried anddirectly revived, induced and applied to wounds (R2LC_pLAB112_Luc, n=4,R2LC_pLAB112_LrCK1.4, n=5).

FIGS. 24A & 24B. Wound size (FIG. 24A) and wound exposure (FIG. 24B)over time in healthy mice. Wound size was measured daily from imageswith a scale included. The change is due to time and treatment and thereis a trend towards decreased wound size by CXCL12 1α in pH of 6.35compared to suspension with pH 7.35 (p=0.07) (pH 7.35; n=8, pH 6.35;n=5, pH 5.35; n=4). One-way ANOVA, Bonferroni compare all columns.

FIGS. 25A & 25B. Wound size (FIG. 25A) and wound exposure (FIG. 25B)over time in healthy mice. Wound size measured daily from images with ascale included. The observed change was only due to time and did notdiffer between the two different bacterial suspensions(R2LC_pLAB112_Luc; n=4, R2LC_pLAB112_LrCK1; n=5). Student's two-tailedunpaired t-test.

FIGS. 26A, 26B, 26C. Measurements of CXCL12 1α levels sections of theskin just next to the wound two days post wound induction in dermis(FIG. 26A), epidermis (FIG. 26B) and hair follicles (FIG. 26C) where thewounds were treated with Lactobacillus reuteri R2LC_pLAB112_LrCK1 at OD0.5, 1.0, and OD 1.25. One-way ANOVA, Bonferroni compare all columns.

FIGS. 27A & 27B. Measurements of density of F4/80⁺ macrophages in dermis(FIG. 27A) and epidermis (FIG. 27B) in the skin next to the wound twodays following wound induction in control wounds and wounds treated withLactobacillus reuteri R2LC_pLAB112_LrCK1 at OD 0.5, 1.0 and OD 1.25.(Control, n=15; 0.5, n=10, OD 1.0, n=4; OD 1.25, n=5). One-way ANOVA,Bonferroni compare all columns.

FIGS. 28A, 28B & 28C. Time to wound healing in healthy mice. Wounds weretreated with Lactococcus Lactis transformed with pLAB112(L.L_pLAB112_LrCK1) or control Lactococcus Lactis. Time to 50% (FIG.28A), 75% (FIG. 28B) or complete (100%) (FIG. 28C) healed wound surface,n=5 both groups. Student's two-tailed unpaired t-test.

FIGS. 29A & 29B. Wound size (FIG. 29A) and wound exposure (FIG. 29B)over time in healthy mice. Wound size measured daily from images with ascale included, n=5 both groups. The change is due to time and treatmentand wound size is decreased by L.L_pLAB112_LrCK1 at d1 to d4 compared tocontrol Lactococcus Lactis. Student's two-tailed unpaired t-test.

FIGS. 30A, 30B & 30C. Time to wound healing in healthy mice treated withrecombinant chemokines for one hour. Time to 50% (FIG. 30A), 75% (FIG.30B) or complete (100%) (FIG. 30C) healed wound surface (Control; n=11,mCXCL12 1α; n=6, mCXCL17; n=8, mYm1; n=9). One-way ANOVA, Bonferronicompare all columns.

FIGS. 31A & 31B. Wound size (FIG. 31A) and wound exposure (FIG. 31B)over time in healthy mice treated with recombinant chemokines for onehour. Wound size measured daily from images with a scale included(Control; n=11, mCXCL12 1α; n=6, mCXCL17; n=8, mYm1; n=9). The change isdue to time and treatment and wound size is decreased by CXCL12 1α,CXCL17 and Ym1 compared to Control. One-way ANOVA, Bonferroni compareall columns.

FIG. 32. Wound closure during the 24 first hours in healthy mice with noor different treatments. (No treatment, n=15; 0.2 μg CXCL12 1α, n=4; 0.6μg CXCL12 1α, n=5; 1.0 μg CXCL12 1α, n=4; 0.2 μg CXCL12 1α 1 hr, n=6;0.2 μg CXCL17 1 hr, n=9, 0.2 μg Ym1 1 hr, n=9; R2LC_pLAB112_Luc OD 0.5,n=4; R2LC_pLAB112_LrCK1.4 OD 0.2, n=4; R2LC_pLAB112_LrCK1.4 OD 0.5,n=10, R2LC_pLAB112_LrCK1.4 OD 1.0, n=4; R2LC_pLAB112_LrCK1.4 OD 1.25;n=5, Freeze-dried R2LC_pLAB112_Luc, n=4, Freeze-driedR2LC_pLAB112_LrCK1.4, n=5, R2LC_pLAB112_Luc supernatant; n=4,R2LC_pLAB112_LrCK1.4 supernatant, n=5, pCTR n=8; pCXCL12, n=9). Nostatistical analyses have been performed on this dataset.

FIGS. 33A & 33B. Assessment of DSS-induced disease activity daily (FIG.33A) and total disease burden, day 1-7 (FIG. 33B). Similar ameliorationof DSS-induced colitis disease activity by treatment with Lactobacillusreuteri pLAB112_Luc and pLAB112_LrCK1.4 (DSS+Vehicle; n=5,DSS+R2LC_pLAB112_Luc; n=6, DSS+R2LC_pLAB112_LrCK1.4; n=7) as compared tothe control group treated with vehicle, One-way ANOVA, Bonferronicompare all columns.

FIGS. 34A & 34B. Assessment of DSS-induced disease activity daily (FIG.34A) and total disease burden, day 1-8 (FIG. 34B). Disease activity wasassessed measuring relevant clinical symptoms as described earlier (Ref.16). Arrow indicates start of treatment. Amelioration of DSS-inducedcolitis disease activity by treatment with Lactobacillus reuteripLAB112_LrCK1.4 compared to treatment with pLAB112_Luc(DSS+R2LC_pLAB112_Luc; n=6, DSS+R2LC_pLAB112_LrCK1.4; n=6), Student'stwo-tailed unpaired t-test.

FIGS. 35A, 35B & 35C. Representative images of full thickness skinwounds (5 mm diameter) induced in healthy mice at time 0 and after 24hours with no treatment, with R2LC Luc or R2LC LrCK1. Images are takenwith a scale included in anesthetized mice.

EXAMPLES Materials and Methods

Gene Construct Design and Production

The plasmid backbone pLAB112 (equal to pSIP411; Refs. 11 and 15; TableI) was provided by Professor Lars Axelsson (Norwegian Food ResearchInstitute). Lactococcus lactis MG1363 bacteria was transformed withpLAB112 and expanded for 24 hours. The plasmid was then purified and theDNA product was verified on a gel.

TABLE I Main features of pSIP411/pLAB112 Feature Positions (SEQ ID NO:20) Replication determinant (replicon region)  260-2010 ermB(erythromycin resistance marker) 2342-2840 P_(spp)IP (induciblepromoter) 3139-3290 sppK (histidine protein kinase) 3305-4647 sppR(response regulator) 4653-5396 gusA (beta-glucuronidase) 5853-7658P_(orf)X (inducible promoter) 5689-5835 Transcriptional terminators129-155; 5428-5460; 5602-5624 Multicloning sites 1-35; 5851-5856;7662-7673

The sequence for murine CXCL12-1α was optimized for translation inLactobacillus reuteri by Stefan Roos at the Swedish University ofAgricultural Sciences (SLU) using DNA2.0 (Menlo Park, Calif., USA). Theoptimized sequence (SEQ ID NO: 1) was synthesized by DNA 2.0 in plasmidvector pJ204. The sequences for human CXCL12-1α, murine CXCL17, humanCXCL17, murine Ym1 and human Ym1 were optimized for translation inLactobacillus reuteri by Stefan Roos at SLU using GenScript (Piscataway,N.J., USA). The optimized sequences are shown as SEQ ID NO: 4 (humanCXCL12-1α); SEQ ID NO: 7 (murine CXCL17); SEQ ID NO: 10 (human CXCL17);SEQ ID NO: 13 (murine Ym1); and SEQ ID NO: 16 (human Ym1).

Primers were designed to detect the insert (hCXCL12opt), 171 bp inpLAB112:

5′GCAGCCTTAACAGTCGGCACCT3′; (SEQ ID NO: 22) 5′ACGTGCAACAATCTGCAAAGCAC3′.(SEQ ID NO: 23)

The ends of the insert were also optimized for continuing the molecularprocessing so the insert would fit in the new vector pLAB112. Theoptimized mCXCL12opt sequence was delivered in a plasmid PJ204. E. coliPK401 was transformed with pJ204. Plasmids (pLAB112 and pJ204) werecleaved with the restriction enzymes XhoI and NcoI in NEB2 buffer. Thefragment mCXCL12opt was then purified on a gel. The mCXCL12opt insertwas then ligated into the pLAB112 vector using T4 DNA ligase, resultingin the construct mLrCK1. The insert construct in the pLAB112 vector wasverified by PCR. The construct was then verified by sequence analysis(Macrogen). Finally Lactobacillus reuteri strain R2LC and DSM 20016 wastransformed with mLrCK1 and two R2LC clones (4 and 7) positive for theconstruct were collected and the plasmid mLrCK1 (now mLrCK1.4 andmLrCK1.7) from these colonies were again verified by sequence analysis(Macrogen).

The plasmids hLrCK1, mLrCK2, hLrCK2, mLrMP1 and hLrMP2 were produced inan analogous way following the same protocol and procedure (See Table IIbelow).

TABLE II Overview of plasmids Plasmid Description pLAB112 Identical withpSIP411 (Ref. 15 and SEQ ID NO: 20) mLrCK1 pLAB112 with optimizedmCXCL12-1α insert mLrCK1.4 mLrCK1 from transformed Lactobacillus reuteriR2LC clone 4 mLrCK1.7 mLrCK1 from transformed Lactobacillus reuteri R2LCclone 7 hLrCK1 pLAB112 with optimized hCXCL12-1α insert mLrCK2 pLAB112with optimized mCXCL17 insert hLrCK2 pLAB112 with optimized hCXCL17insert hLrMP1 pLAB112 with optimized human Ym1 insert mLrMP2 pLAB112with optimized murine Ym1 insert pLAB112_Luc pLAB112 with luciferaseinsert

In Vitro Analysis of Plasmid Expression

Lactobacillus reuteri R2LC pLAB112_Luc cultured overnight, re-inoculatedand grown to OD 0.5 were plated (200 μl/well) on a 96 well plate orimmediately resuspended from freeze-dried formulation. Luminescenceintensity was determined using non-invasive bioimaging (IVIS Spectrum,Perkin Elmer). A baseline image at time 0 was acquired. Then activationpeptide SppIP (50 ng/ml) and D-Luciferin (150 μg/ml) was addedimmediately after. The plate was then imaged at 5 minutes and then every30^(th) minute for 1400 minutes. Data was quantified using Living Image3.1 software (Perkin Elmer) and imaging parameters were maintained forcomparative analysis. Radiance was considered proportional to plasmidexpression.

Animals

Experiments were approved by Uppsala Regional Laboratory Animal EthicalCommittee. Mice, C57Bl/6 (Taconic) and CX3CR1^(+/GFP) on C57Bl/6background (originally from The Jackson Laboratory) were used. Animalshad free access to water and chow throughout experiments.

Wound Induction

Mice were anesthetized (1-3% isoflurane, 200 ml/min) and hair wasremoved on the hind limb by shaving and then by 1 min application ofhair removal cream (Veet®) that were rinsed off with water. A sterilepunch biopsy needle (5 mm diameter) was used to induce full-thickness(epidermis, dermis and subcutis) wounds. Local topical analgesic (Emblacream) was applied daily for the first 5 days.

Topical Wound Treatments

Wounds were treated daily with either 25 μl saline, Lactobacillusreuteri R2LC pLAB112_Luc or R2LC pLAB112_LrCK1. Bacteria was culturedovernight, re-inoculated and grown to OD 0.5, preactivated 5 min priorto application with activation peptide SppIP (50 ng/ml) and addedtopically to the middle of the wound surface. For dosing experimentswounds were treated daily for two days with either 25 μl saline orLactobacillus reuteri R2LC pLAB112_LrCK1 re-inoculated from overnightculture and grown to OD 0.5, preactivated 5 min prior to applicationwith activation peptide SppIP (50 ng/ml) and added topically to themiddle of the wound surface at concentrations of OD 0.2, 0.5, 1.0 or1.25. For comparative experiments with the respective proteins woundswere treated daily with either 10 μl saline or murine CXCL12, CXCL17 orYm1 (total of 200 ng protein in 60 μl saline given in 10 min intervalsfor one hour). For a dose escalation study of CXCL12 200 ng, 600 ng or 1μg was added to the wound in 10 μl saline at one time point once perday.

In Vivo Analysis of Plasmid Expression

Lactobacillus reuteri R2LC pLAB112_Luc were cultured overnight,re-inoculated and grown to OD 0.5. Luminescence intensity was determinedusing non-invasive bioimaging (IVIS Spectrum, Perkin Elmer). A baselineimage at time 0 was acquired. Then 25 μl Lactobacillus reuteri R2LCpLAB112_Luc was added in the middle of the wound. Bacteria waspreactivated 5 min prior to application with activation peptide SppIP(50 ng/ml) and D-Luciferin (150 μg/ml). Mice were the imaged every15^(th) minute for 270 minutes. Data was quantified using Living Image3.1 software (Perkin Elmer) and imaging parameters were maintained forcomparative analysis. Radiance was considered proportional to plasmidexpression.

Wound Size and Appearance Monitoring

The size and appearance of the wounds were monitored daily inanesthetized mice (1-3% isoflurane, 200 ml/min) by acquisition ofconventional photos. A scale was included in the image at acquisitionand wound size was analyzed using ImageJ (Free software from NIH).Wounds were considered healed when <0.5 mm² in size.

Cutaneous Blood Flow Monitoring

Blood flow in the whole hind limb with the healing wound was measured inanesthetized (1-3% isoflurane, 200 ml/min) mice using noninvasive LaserSpeckle Contrast Analysis and data was analyzed, PIMSoft 3 (Perimed).Limbs (Frame 1.4×1.4 cm) were imaged for 2 minutes at 10 images/s withaveraging by 20. Data is expressed in perfusion units (PFU).

Reduction of Perfusion

Mice were anesthetized (1-3% isoflurane, 200 ml/min) and hind limbischemia was induced by ligation and excision of the femoral arteryabove the superficial epigastric artery branch.

Induction of Hyperglycemia

A single dose of alloxan monohydrate (8 mg/ml, 1 μl/g body weight)immediately dissolved in sterile saline was injected in the tail vein.Blood glucose and body weight was monitored daily throughout theexperiment. Hyperglycemia was defined as blood glucose>16.7 mmol/l.

Statistical Analysis

Data are presented as mean±SEM. Two-Way ANOVA with Bonferroni compareall columns post hoc test was used analyzing the healing process overtime. One-Way ANOVA with Bonferroni compare all columns post hoc testwas used analyzing the healing process at one time point in groups ofn>2 and Students two-tailed unpaired t-test was used analyzing thehealing process at one time point when n=2. p<0.05 was consideredstatistically significant.

Example 1: Growth of Bacteria Transformed with Plasmid LrCK1

Lactococcus lactis with mLrCK1 cultured overnight, re-inoculated andgrown to OD 0.3 or 0.5 showed no growth impairment when the activationpeptide SppIP (SEQ ID NO: 19) were added at either 10 or 50 ng/ml.During these growth experiments pH was measured and the lowering wasmost accentuated in the growth phase and then stabilized around pH 6.7when grown is Mes-medium (FIGS. 1A & 1B). (pH of skin=5,5, pH inwounds=7.15-8.9 where alkaline pH correlates with lower healing rate(Ref. 14))

Example 2: Expression of Plasmid pLAB112 Luc

In vitro expression of plasmid pLAB112_Luc in Lactobacillus reuteri R2LCre-inoculated and grown for 2 hours from overnight culture remained highfor more than 600 minutes (10 h.). There was no leakage/expression fromplasmids not activated with activation peptide SppIP (FIG. 2).

When Lactobacillus reuteri R2LC with pLAB112_Luc re-inoculated and grownfor 2 hours from overnight culture were placed in 1 day old cutaneousfull thickness wounds of anesthetized mice, bacteria was restricted tothe wound site and plasmid expression was high for the first hour butsignal was detected for more than 4 hours (FIG. 3).

Example 3: Improved Wound Healing in Healthy Mice

Wounds were monitored daily during the healing process. In healthy micedaily single application of Lactobacillus reuteri R2LC_pLAB112_mLrCK1.4reduced time to both 75% wound surface closure and to complete (100%)wound closure compared to control mice where nothing was applied to thewound and to mice where control Lactobacillus reuteri R2LC (pLAB112_Luc)was applied daily (FIGS. 4A, 4B & 4C). The effect of Lactobacillusreuteri R2LC_pLAB112_mLrCK1.4 on wound healing was most prominent duringthe first days post wound induction. Wound size was then further reducedby daily application (one and two days post wound induction) ofLactobacillus reuteri R2LC_pLAB112_mLrCK1.4 when compared to controlmice where nothing was applied to the wound. The total wound exposuremeasured as area under curve was also reduced in this group compared tocontrol mice where nothing was applied to the wound (FIGS. 5A & 5B).FIGS. 35A, 35B & 35C show representative images of full thickness skinwounds (5 mm diameter) induced in healthy mice at time 0 and after 24hours with no treatment, with R2LC Luc or R2LC LrCK1.

Example 4: Improved Wound Healing in Healthy Mice Having Impaired TissuePerfusion

Cutaneous perfusion was reduced by 50% at the day of wound induction byligation of the femoral artery in the limb where the wound was induced(FIGS. 6A & 6B and Table III). In mice with ischemia, daily singleapplication of Lactobacillus reuteri R2LC_pLAB112_mLrCK1.4 resulted inreduced time to both 50% and 75% wound surface closure compared tocontrol mice where nothing was applied to the wound as well as to micewhere control Lactobacillus reuteri R2LC (pLAB112_Luc) was applied daily(FIGS. 7A, 7B & 7C). Also in mice with reduced cutaneous perfusion theeffect of Lactobacillus reuteri R2LC_pLAB112_mLrCK1.4 on wound healingwas most prominent during the first days post wound induction, and woundsize were reduced by daily application of Lactobacillus reuteri

R2LC_pLAB112_mLrCK1.4 at one and two days post wound induction comparedto control mice where nothing was applied to the wound. The total woundexposure was also reduced in this group compared to control mice wherenothing was applied to the wound (FIGS. 8A & 8B).

TABLE III Basal skin perfusion measured by Laser Speckle ContrastAnalysis in anesthetized mice. Data is expressed as Mean ± SEM inperfusion units (PFU), n = 4 all groups. Healthy Ischemic Reduction (%)Control 62.5 ± 4.3 34.0 ± 1.8 46 R2LC_pLAB112_Luc 57.3 ± 2.7 31.3 ± 1.146 R2LC_pLAB112_LrCK1.4 65.0 ± 7.2 30.8 ± 0.4 52

Example 5: Improved Wound Healing in Hyperglycemic Mice

Mice were rendered diabetic using alloxan, where after they remainedhyperglycemic (>16.7 mmol/l) during the process of wound healing and didnot lose weight (FIGS. 9A & 9B). In mice with diabetes, daily singleapplication of Lactobacillus reuteri R2LC_pLAB112_LrCK1.4 reduced timeto 75% wound surface closure compared to control mice where nothing wasapplied to the wound and to mice where control Lactobacillus reuteriR2LC (pLAB112_Luc) was applied daily (FIGS. 10A, 10B & 10C). There was atrend (p=0.08) towards reduced wound exposure in diabetic mice by dailyapplication of Lactobacillus reuteri R2LC_pLAB112_mLrCK1.4 compared todaily application of Lactobacillus reuteri R2LC with Luc and controlmice where nothing was applied to the wound (FIGS. 11A & 11B).

Example 6: CXCL12 Dermal Overexpression in the Wound Edge DermisTransfection with Plasmid Encoding CXCL12

Plasmids were constructed on the pVAX1 backbone with CMV promoter (SEQID NO: 24) (V260-20, Invitrogen, Waltham, Mass., USA), and either insert-copGFP-T2A-Luc2- referred to as pCTR (SEQ ID NO: 25) or-CXCL12-P2A-copGFP-T2A-Luc2- referred to as pCXCL12 (SEQ ID NO:26) wasintroduced as previously described (Ref. 18). The secretion sequence forCXCL12 was substituted for the murine IgG secretory sequence. Thus, pCTRplasmids encode GFP (Green Fluorescent Protein) and luciferase but nochemokines. Plasmids (40 μg in a total volume of 100 μl saline) wereinjected in the dermis in four locations in the wound edge. Transgeneexpression was measured over time based on luciferase activity followingintraperitoneal injection of D-Luciferin (150 mg/kg, #122796, PerkinElmer, Waltham, Mass., USA) 10 min prior to anesthesia and imageacquisition using a bioimaging device (IVIS Spectrum, Perkin Elmer).Data was quantified using Living Image 3.1 software (Perkin Elmer) andimaging parameters were maintained for comparative analysis. Settingswere also maintained selecting region of interest where thecontralateral reference area was subtracted. Radiance was consideredproportional to plasmid expression.

Plasmid expression from the dermis in the wound edge was measured usingnon-invasive bioimaging and correlated to light produced by theluciferase enzyme encoded by the plasmids equivalent to the expressionof CXCL12. Expression peaked on day 2 and then declined as the wound wasclosing and the dermis reconstituted (FIG. 13). Overexpression of CXCL12did not result in accelerated complete wound healing but lead to shortertime to closure of 75% of the wound surface as compared to pCTR (FIGS.14A, 14B & 14C). Wound surface was decreased by pCXCL12 dermalexpression as compared to pCTR day 4-6 post wound induction and dermistransfection (FIGS. 15A & 15B). These results demonstrate that withCXCL12 delivered to the wound with this system there is not a dramaticeffect the 24 first hours but rather a smaller effect at the later timepoints.

Example 7: Dose-Response Lactobacillus reuteri of Topical Treatment withLuc and LrCK1

Lactobacillus was reinoculated from overnight culture and grown to OD0.5 and then diluted or concentrated to OD 0.2, 0.5, 1.0 and 1.25 inMRS. The four different concentrations were diluted tenfold to 10⁻⁹ and10 μl of every sample was plated on MRS agar with erythromycin andcultured in an anaerobic chamber overnight in at 37° C., 5% carbondioxide overnight. Colonies on the plates were counted and concentrationexpressed as colony forming units per ml (CFU/ml).

For dosing experiments wounds were treated daily for two days witheither 25 μl saline or Lactobacillus reuteri R2LC pLAB112_LrCK1.4re-inoculated from overnight culture and grown to OD 0.5, preactivated 5min prior to application with activation peptide SppIP (50 ng/ml) andadded topically to the middle of the wound surface at concentrations ofOD 0.2, 0.5, 1.0 or 1.25. In 25 μl OD of 0.5 there are 5×10⁷ bacteria(2×10⁹ cfu/ml) meaning a dose span of 1000 times.

Bacterial concentration was measured by optical density and colonyforming units per ml are displayed in FIG. 16. The lowest dose (OD 0.2equals 2×10⁷ bacteria) of R2LC_pLAB112_LrCK1.4 cultured and activated asbefore administered to the wound resulted in the smallest wound sizeafter 24 hours and all four different concentrations resulted insignificantly accelerated wound closure at 24 and 48 hours post woundinduction (FIG. 17A) and thus resulted in decreased wound exposure tothe first 48 hours as compare to wounds receiving no treatment (FIG.17B). These results indicate that administration of a dose that is 10³higher (OD 1.25 equals 1×10¹⁰ bacteria) than the lowest dose giving thegreatest effect also significantly accelerates wound healing the first48 hours as compared to wounds receiving no treatment and to the sameextent as the dose giving maximal wound closure. No signs of inducedinflammation or other negative side effects were observed for woundsgiven the highest dose. The data show that even a low dose ofLactobacillus reuteri R2LC_LrCK1 accelerates wound healing.

Example 8: Dose Escalation of mCXCL12 1α Protein as a Topical Treatment

To investigate the effects of the dose of the mCXCL12 1α administered tothe wound surface 0.2 μg, 0.6 μg or 1 μg mCXCL12 1α (RnD Systems) wasdelivered to the wounds daily for two days in 10 μl saline. Theadministration was once per day.

Delivery of the mCXCL12 1α daily at one single time point per day didnot accelerate wound healing for the first two days as compared to notreatment (FIGS. 18A & 18B). These data shows that it is the continuousdelivery of the CXCL12 1α that causes the accelerated wound healingsince a total 0.2 μg mCXCL12 1α given every day for one hour in 10 minintervals accelerated healing the first 48 hours (FIGS. 18A, 18B, 19A &19B).

Example 9: Re-Epithelialization Assay in Human Skin Biopsies

Sterile normal human skin was obtained from healthy white women havingroutine breast reduction at Uppsala University Hospital giving consentfor donation. Samples were covered with physiological DMEM supplementedwith 2% bovine calf serum (Hyclone®, HyClone Laboratories, Logan USA)and transported to the laboratory under sterile conditions.

As previously described (Ref. 17), the subcutis was removed andremaining dermis and epidermis was cut using a 6 mm skin biopsy punch(Integra Miltex, York, Pa., USA) and sterile scissors. In the center ofeach 6 mm diameter skin disc the epidermis was removed using a 3 mm skinbiopsy punch and sterile scissors. Samples were then placed one by onein a sterile 24 well plate with the epidermal side up. All culture media(DMEM) was supplemented with BSA, 2 or 10% and antibiotics (erythromycinSigma Aldrich, Buchs, Swizerland at 10 μg/ml). To maintain the nutrientson the dermal side i.e. nutrients at the highest concentration on thedermal side of the skin, 0.5 ml medium was added to each well and mediumwas changed daily. At the same time as the change of medium 10⁶ in 10 μlMRS Lactobacilus reuteri R2LC_Luc or Lactobacilus reuteri R2LC_LrCK1were placed in the middle of the epidermal wound in the floating skindiscs. The bacteria was inoculated and grown in MRS for 2-4 hours to bein the exponential phase. Samples were incubated at 37° C., 5% carbondioxide, and 95% humidity for 14 days.

The specimens were cut through the middle and one half was fixatedovernight in 4% formaldehyde, pH 7.38 and dehydrated through anethanol-xylene series to finally be embedded in paraffin. Cross-sections(10 μm) starting from the part being at the center of the specimens,were mounted, deparaffinized, rehydrated, and stained with hematoxylinand eosin. Images were captured using Leica Leits Dmrb with a LeicaDFC420 C camera and Plan Fluot 40×0.7 NA objective. Re-epitelializationor epidermis sleeve length was measured in images using ImageJ (NIH).

Adding Lactobacillus to the skin discs in culture lowered the pH of theculture medium when measured after 24 hours (FIG. 20A). The epidermis onthe edges of the induced wound in the skin discs was proliferating tocover the exposed dermis when 10% FCS was present in the culture mediumand there was almost no proliferation when skin discs were cultured inmedium supplied with 2% FCS after 14 days in culture (FIG. 20B). Nodetrimental effects were macroscopically detected in the skin discstreated with R2LC pLAB112 Luc or R2LC pLAB112_LrCK1 and increasedre-epithelialization was measured on wounds where the skin discs weretreated with R2LC pLAB112_LrCK1 for 14 days (FIG. 20B).

Example 10: Functionality of Bacteria after Freeze-Drying and Revival

Different protocols and 35 different formulations for freeze-drying weretested and viability was measured for up to two months. Also a largerbatch of freeze-dried Lactobacillus reuteri was produced in settingsidentical to large scale industrialized production and in accordancewith good manufacturing practice. The freeze-dried samples from thisbatch have been analyzed for viability after storing for up to twomonths in temperatures ranging from −20 to 40° C. Freeze-dried bacteriawere revived by adding equivalent volume of water or MRS medium withSppIP (50 ng/ml) and then analyzed immediately for expression in vitroand in vivo by plating them in a 96 well plate or applying them directlyon 1 day old wounds as described above.

With the most promising formulation, viability was stable from directlyafter freeze-drying to analysis at two months measured on samples storedat +4° C. The viability was well within range of what is acceptable offreeze-dried bacteria currently being sold as dietary supplements.Measuring the plasmid expression in freeze-dried Lactobacillus reuteriR2LC_pLAB112_Luc directly after resuscitation showed immediate inductionof expression, which peaked at 450 minutes and then declined (FIG. 21).After 24 hours (1440 minutes) there was no expression and no alivebacteria. When freeze-dried Lactobacillus reuteri R2LC_pLAB112_Luc wererevived, induced with 50 ng/ml (SpplP) and immediately placed oncutaneous wounds of mice (5×10⁷ per 25 μl), expression directlyincreased and was high for about one hour (FIG. 22) in a similar patternas was seen when adding fresh bacteria in growth phase in solution (FIG.3).

The effect on wound healing was tested where the freeze-dried bacteria(5×10⁷ per 25 μl) was again revived, induced and immediately placed oncutaneous wounds of mice. The wounds were monitored every day for twodays and the wounds treated with Lactobacillus reuteriR2LC_pLAB112_LrCK1 showed accelerated healing compared to wounds treatedwith Lactobacillus reuteri R2LC_pLAB112_Luc (FIGS. 23A & 23B) even withthis protocol. These data show that the Lactobacillus reuteriR2LC_pLAB112 does not have to be precultured to the exponential growthphase in order to produce and deliver enough CXCL12 to accelerate woundhealing in vivo.

Example 11: pH Dependent Effects of Chemokine Signaling

Chemokines can appear as monomers, dimers or multimers either withitself or interacting with other chemokines (Ref. 22). The differentcombinations and conformations induce different receptor signaling andthus different cell responses (Ref. 34). This is a new and unexploredarea and the combination of possibilities is dependent on the localtissue microenvironment. Also local pH impacts on local macrophagefunction (Ref. 23).

For studies of pH dependent effects of chemokine potency, 0.2 μg CXCL121α was applied to wounds in 10 μl saline with pH 7.35, 6.35 or 5.35daily for two days.

Altering the pH in the buffer containing the chemokines had an effect onthe healing pattern of the treated wounds and there was a trend towardssmaller wound size one day post wound induction when the CXCL12 weresuspended in saline with pH of 6.35 compared to when the CXCL12 weresuspended in saline with pH of 7.35 (p=0.07) (FIGS. 24A & 24B). Thesedata indicate that a pH of 6.35 potentiates the effect of recombinantCXCL12 applied to the wound surface in the aspect of inducingaccelerated wound healing.

Example 12: Importance of Bacterial on Site Chemokine Delivery to theWound Surface for Effect

For wound treatment with fresh supernatants Lactobacillus reuteriR2LC_pLAB112_Luc and R2LC_pLAB112_LrCK1 were inoculated in 10 ml MRS in37° C. and grown to OD 0.5, centrifuged (>2000 rpm, 5 minutes),resuspended in 1 ml MRS, activated (SppIP, 50 ng/ml) and grown for 4hours. Samples were then centrifuged (>2000 rpm, 5 minutes) and thesupernatant was saved. 25 μl of this supernatant was then applied towounds once daily for two days.

The importance of bacterial delivery of CXCL12 1α directly to the woundsurface by the Lactobacillus reuteri R2LC_pLAB112_LrCK1 was demonstratedin a model where fresh supernatants from induced Lactobacillus reuteriwere added to the wounds following wound induction every day for twodays. There was no difference in wound size or total wound exposure(p=0.2595) of treatment with fresh supernatants from Lactobacillusreuteri R2LC_pLAB112_Luc or R2LC_pLAB112_LrCK1 (FIGS. 25A & 25B).

Example 13: Lactobacillus Delivered CXCL12 Increases Levels of CXCL12 inthe Skin Surrounding the Wound

For quantitative analysis the skin surrounding the wound (0-100 μm fromthe wound) was removed on the last day of experiments and snap frozen inliquid nitrogen and sectioned (10 μm). After fixation in ice coldmethanol (10 min) and permeabilization in 0.5% Triton-X (15 min) tissueswere incubated with antibodies targeting CXCL12 1α (polyclonal, Abcam)and macrophage antigen F4/80 (clone BM8, eBioscience) washed andincubated with matching secondary antibodies conjugated to AlexaFluor488 and Nordic Lights 557 (Invitrogen). Tissues were finally washedand mounted (Fluoromount, #0100-10, Southern Biotech, Birmingham, Ala.,USA) before imaging using a line-scanning confocal microscope (Zeiss LSM5 Live, with a piezo motor-controlled WPlanApo 40×/1.0 with 0.5 opticalzoom, Zeiss, Oberkochen, Germany). Protein levels and macrophages werequantified in images using ImageJ (NIH) and IMARIS software 8.2(Bitplane, Zurich, Switzerland). Microscope settings were maintainedduring acquisition to allow comparison. Values for CXCL12 1αmeasurements are presented as mean fluorescent intensity (MFI).

Treatment of wounds once daily for two days with Lactobacillus reuteriR2LC_pLAB112_LrCK1 in different doses resulted in increased skin tissuelevels of CXCL12 1α in the skin just next to the wound compared to inthe skin next to wounds receiving no treatment (FIGS. 26A, 26B & 26C)and this was true for both dermis, epidermis and in hair follicles.

Example 14: Lactobacillus Delivered CXCL12 Increases Macrophages in theSkin Surrounding the Wound

Treatment of wounds once daily for two days with Lactobacillus reuteriR2LC_pLAB112_LrCK1 in different doses resulted in increased density ofF4/80+ macrophages in dermis just next to the wound two days post woundinduction when Lactobacillus reuteri R2LC_pLAB112_LrCK1 at OD 0.2 and OD0.5 were applied to the wound compared to the dermis next to woundsreceiving no treatment (FIG. 27A). F4/80+ macrophages were increased inthe epidermis next to the wound two days post wound induction whenLactobacillus reuteri R2LC_pLAB112_LrCK1 were given to the wound surfaceat OD 1.25 as compared to the epidermis next to wounds receiving notreatment (FIG. 27B).

Example 15: Verification of Effect on Acceleration of Wound HealingUsing Lactococcus Lactis

To show that the local and continuous delivery of the specific chemokineproduced by the bacteria is important for the mechanism irrespectivelyof bacterial strain, another strain was used to produce and deliver thechemokine directly to the wound surface, Lactococcus Lactis wastransformed with pLAB112 (mLrCK1). Bacteria were applied once daily tofull thickness wounds in healthy mice following the same protocol asdescribed for treatment with using Lactobacuillus reuteri.

There is a clear trend that mCXCL12 1α delivery accelerates woundclosure (FIGS. 28A, 28B & 28C) and reduces wound size and exposure inthis model (FIGS. 29A & 29B).

Example 16: Moderate Effects on Time to Wound Closure by Treatment withmCXCL12 1α, mCXCL17 and mYm1 Delivered as Recombinant Proteins

To show that the mode of delivery and continuous protein productionenabled by the lactic acid bacteria is important for the mechanism,murine recombinant mCXCL12 1α, mCXCL17, mYm1 (total of 200 ng in 60 μl,all RnD Systems) or saline (10 μl) as control was delivered to the woundonce daily every 10th minute for one hour.

For mCXCL12 1α, delivery of 30 ng into the peritoneal cavity inducessignificantly increased recruitment of immune cells in 3 hours, why 200ng to an area of 25 μm² is to be considered a high dose.

It is likely that the high protease activity in the wound degrades thechemokines when given as recombinant protein at one single time point,and thus the de novo production by the bacteria is required for theprotein to enhance wound closure. In addition, the lactic acid bacteriamight also provide a beneficial local environment for wound healing(FIGS. 1B, 4A, 4B, 4C, 5A, 5B, 24A, 24B, 30A, 30B, 30C, 31A and 31B).

Example 17: Comparison of the Effects of Different Treatments on WoundClosure in Healthy Mice

Wound closure during the 24 first hours in healthy mice was analyzed forall the different treatments performed (FIG. 32). It is clear thattreatment with Lactobacillus reuteri R2LC_Luc or low single doses ofCXCL12 1α administered to the wound at one time point affects thehealing during the 24 first hours. Though there is a trend that CXCL121α, CXCL17 and Ym1 delivered to the wound surface every 10^(th) minutefor one hour accelerates the wound closure during the first 24 hours andthis effect is even more clear when the CXCL12 1α is deliveredcontinuously for one hour to the wound surface by Lactobacillus reuteriR2LC_LrCK1. Delivering the CXCL12 via dermal overexpression rather havea detrimental effect on 24 hour wound closure.

Example 18: Acceleration of Wound Healing Also on Mucosal Surfaces byLactobacillus reuteri with pLAB112_mLrCK1.4

To test if the local continuous delivery of CXCL12 to a wounded surfaceworks through a global mechanism on both skin epithelium and intestinalepithelium, two experimental protocols of DSS-induced colitis was used.DSS (dextran sulfate sodium) is known to induce wounds in the mucosalsurface of the colon (Ref. 16).

For the first protocol, mice were treated with Lactobacillus reuteri bygavage (1 ml OD 0.5 spun and resuspended in 0.1 ml) once daily for 14days while DSS was given in the drinking water day 7-14. Since thisstrain of Lactobacillus reuteri colonizes in the colon using thisprotocol the aim is to assess if presence of Lactobacillus reuteripLAB112_mLrCK1 in the colon is beneficial as compared to Lactobacillusreuteri pLAB112_Luc when colitis is induced.

The second protocol aimed at treating manifest colitis, and mice weregiven DSS in the drinking water day 1-8 while receiving Lactobacillusreuteri by gavage three times daily at day 5-8.

The severity of colitis was assessed daily on the basis of clinicalparameters including weight loss, stool consistency and blood content,and presented as Disease Activity Index (DAI), a scoring methoddescribed in detail by Cooper and coworkers (Ref. 16).

There was similar amelioration of DSS-induced colitis disease activityby pretreatment with Lactobacillus reuteri pLAB112_Luc andpLAB112_LrCK1.4 (FIGS. 33A & 33B) indicating the effect is only due tothe Lactobacillus reuteri.

In contrast, disease development was ameliorated when Lactobacillusreuteri pLAB112_LrCK1.4 was administered to colitic mice which was notobserved for treatment with pLAB112_Luc (FIGS. 34A & 34B) indicatingeffect of the delivered chemokine.

REFERENCES

-   1. Demidova-Rice T N, Hamblin M R and Herman I M. Acute and impaired    wound healing: pathophysiology and current methods for drug    delivery, part 1: normal and chronic wounds: biology, causes, and    approaches to care. Advances in skin & wound care. 2012; 25:304-14.-   2. Demidova-Rice T N, Hamblin M R and Herman I M. Acute and impaired    wound healing: pathophysiology and current methods for drug    delivery, part 2: role of growth factors in normal and pathological    wound healing: therapeutic potential and methods of delivery.    Advances in skin & wound care. 2012; 25:349-70.-   3. Salcedo R, Wasserman K, Young H A, Grimm M C, Howard O M Z, Anver    M R, Kleinman H K, Murphy W J and Oppenheim J J. Vascular    Endothelial Growth Factor and Basic Fibroblast Growth Factor Induce    Expression of CXCR4 on Human Endothelial Cells: In Vivo    Neovascularization Induced by Stromal-Derived Factor-1α. The    American Journal of Pathology. 1999; 154:1125-1135.-   4. Hattermann K, Sebens S, Helm O, Schmitt A D, Mentlein R, Mehdorn    H M and Held-Feindt J. Chemokine expression profile of freshly    isolated human glioblastoma-associated macrophages/microglia.    Oncology reports. 2014; 32:270-6.-   5. Badillo A T, Chung S, Zhang L, Zoltick P and Liechty K W.    Lentiviral gene transfer of SDF-1alpha to wounds improves diabetic    wound healing. The Journal of surgical research. 2007; 143:35-42.-   6. Lee W Y, Wang C J, Lin T Y, Hsiao C L and Luo C W. CXCL17, an    orphan chemokine, acts as a novel angiogenic and anti-inflammatory    factor. American journal of physiology Endocrinology and metabolism.    2013; 304:E32-40.-   7. Burkhardt A M, Tai K P, Flores-Guiterrez J P, Vilches-Cisneros N,    Kamdar K, Barbosa-Quintana O, Valle-Rios R, Hevezi P A, Zuñiga J,    Selman M, Ouellette A J and Zlotnik A. CXCL17 Is a Mucosal Chemokine    Elevated in Idiopathic Pulmonary Fibrosis That Exhibits Broad    Antimicrobial Activity. The Journal of Immunology. 2012;    188:6399-6406.-   8. Goren I, Pfeilschifter J and Frank S. Uptake of    Neutrophil-Derived Ym1 Protein Distinguishes Wound Macrophages in    the Absence of Interleukin-4 Signaling in Murine Wound Healing. Am J    Pathol. 2014.-   9. Poutahidis T, Kearney S M, Levkovich T, Qi P, Varian B J, Lakritz    J R, Ibrahim Y M, Chatzigiagkos A, Alm E J and Erdman S E. Microbial    symbionts accelerate wound healing via the neuropeptide hormone    oxytocin. PLoS One. 2013; 8:e78898.-   10. Ramos A N, Cabral M E, Noseda D, Bosch A, Yantorno O M and    Valdez J C. Antipathogenic properties of Lactobacillus plantarum on    Pseudomonas aeruginosa: the potential use of its supernatants in the    treatment of infected chronic wounds. Wound repair and regeneration:    official publication of the Wound Healing Society [and] the European    Tissue Repair Society. 2012; 20:552-62.-   11. Sørvig E, Mathiesen G, Naterstad K, Eijsink V G H and    Axelsson L. High-level, inducible gene expression in Lactobacillus    sakei and Lactobacillus plantarum using versatile expression    vectors. Microbiology. 2005; 151:2439-2449.-   12. Eijsink V G, Axelsson L, Diep D B, Havarstein L S, Holo H and    Nes I F. Production of class II bacteriocins by lactic acid    bacteria; an example of biological warfare and communication.    Antonie van Leeuwenhoek. 2002; 81:639-54.-   13. Gao Z, Tseng C-h, Pei Z and Blaser M J. Molecular analysis of    human forearm superficial skin bacterial biota. Proceedings of the    National Academy of Sciences. 2007; 104:2927-2932.-   14. Gethin G. The significance of surface pH in chronic wounds.    Wounds UK. 2007; 3:52-56.-   15. Sørvig E, Grönqvist S, Naterstad K, Mathiesen G, Eijsink V G,    and Axelsson L. Construction of vectors for inducible gene    expression in Lactobacillus sakei and L plantarum. FEMS Microbiol    Lett. 2003; 229(1):119-126.-   16. Cooper H. S., Murthy S. N., Shah R. S., Sedergran D. J.    Clinicopathologic study of dextran sulfate sodium experimental    murine colitis. Lab. Invest. 1993; 69(2):238-249.-   17. Nyman E, Huss F, Nyman T, Junker J, Kratz G. Hyaluronic acid, an    important factor in the wound healing properties of amniotic fluid:    in vitro studies of re-epithelialisation in human skin wounds. J    Plast Surg Hand Surg. 2013 April; 47(2):89-92.-   18. Vågesjö E, Christoffersson G, Waldén T, Carlsson P, Essand M,    Korsgren O, and Phillipson M. Immunological shielding by induced    recruitment of regulatory T lymphocytes delays rejection of islets    transplanted to muscle. Cell transplantation. 2015; 24(2):263-76.-   19. Böhmer N, König S and Fischer L. A novel manganese    starvation-inducible expression system for Lactobacillus plantarum.    FEMS Microbiol Lett 342 (2013) 37-44.-   20. Duong, T, Miller, M., Barrangou, R., Azcarate-Peril A. and    Klaenhammer T., Construction of vectors for inducible and    constitutive    -   gene expression in Lactobacillus mbt_200 357. Microbial        Biotechnology (2010) 4(3), 357-367.-   21. Sørvig E, Mathiesen G, Naterstad K, Eijsink V G, and Axelsson L.    High-level, inducible gene expression in Lactobacillus sakei and    Lactobacillus plantarum using versatile expression vectors.    Microbiology. 2005 July; 151(Pt 7):2439-49.-   22. Nesmelova I, Sham Y, Gao J, and Mayo K. CXC and C C chemokines    form mixed heterodimers association free energies from molecular    dynamics simulations and experimental correlations. JBC Papers in    Press, Jun. 12, 2008, DOI 10.1074/jbc.M803308200-   23. Bellocq A, Suberville S, Philippe C, Bertrand F, Perez J,    Fouqueray B, Cherqui G, Baud L. Low environmental pH is responsible    for the induction of nitric-oxide synthase in macrophages. Evidence    for involvement of nuclear factor-kappaB activation. J Biol Chem.    1998 Feb. 27; 273(9):5086-92.-   24. Thompson J, Higgins D G, Gibson T J. CLUSTAL W: improving the    sensitivity of progressive multiple sequence alignment through    sequence weighting, position-specific gap penalties and weight    matrix choice    -   (1994) Nucleic Acids Res., 22: 4673-4680.-   25. Myers E and Miller W, Optimal alignments in linear space. (1988)    CABIOS, 4: 11-17.-   26. W. R. Pearson and D. J. Lipman. Improved Tools for Biological    Sequence Analysis (1988) PNAS, 85:2444-2448.-   27. W, R. Pearson (1990), Rapid and sensitive sequence comparison    with FASTP and FASTA. Methods Enzymol., 183: 63-98,-   28. Altschul S F, Madden T L, Schäffer A A, Zhang J, Zhang Z, Miller    W, Lipman D J. (1997) Gapped BLAST and PSI-BLAST: a new generation    of protein database search programs. Nucleic Acids Res., 25:    3389-3402.-   29. Holm L and Sander C. Protein structure comparison by alignment    of distance matrices (1993) J. Mol. Biol., 233: 123-38; 9.-   30. Holm L and Sander C. Dali: a network tool for protein structure    comparison. (1995) Trends Biochem. Sci., 20: 478-480.-   31. Holm L and Sander C. Touring protein fold space with    Dali/FSSP. (1998) Nucleic Acid Res., 26: 316-9).-   32. Massena S, Christoffersson G, Vågesjö E, Seignez C, Gustafsson    K, Binet F, Herrera Hidalgo C, Giraud A, Lomei J, Weström S, Shibuya    M, Claesson-Welsh L, Gerwins P, Welsh M, Kreuger J, Phillipson M.    Identification and characterization of VEGF-A-responsive neutrophils    expressing CD49d, VEGFR1, and CXCR4 in mice and humans. Blood. 2015    Oct. 22; 126(17):2016-26. doi: 10.1182/blood-2015-03-631572. Epub    2015 Aug. 18.-   33. Hatse S, Princen K, Liekens S, Vermeire K, De Clercq E,    Schols D. Fluorescent CXCL12AF647 as a novel probe for    nonradioactive CXCL12/CXCR4 cellular interaction studies.    Cytometry A. 2004 October; 61(2):178-88.-   34. Drury L, Ziarekb J, Gravelc, S, Veldkampb C, Takekoshif T,    Hwangf S, Hevekerc N, Volkmanb B and Dwinella M. Monomeric and    dimeric CXCL12 inhibit metastasis through distinct CXCR4    interactions and signaling pathways. PNAS Oct. 25, 2011, vol. 108,    no. 43, pages 17655-17660

SEQUENCES

TABLE IV Summary of Sequence Listing SEQ ID NO: Description 1.mLrCK1_opt DNA 2. mLrCK1_opt protein 3. mCXCL12 native protein 4.hLrCK1_opt DNA 5. hLrCK1_opt protein 6. hCXCL12 native protein 7.mLrCK2_opt DNA 8. mLrCK2_opt protein 9. mCXCL17 native protein 10.hLrCK2_opt DNA 11. hLrCK2_opt protein 12. hCXCL17 native protein 13.mYm1_opt DNA 14. mYm1 protein 15. mYm1 native protein 16. hYm1_opt DNA17. hYm1 protein 18. hYm1 native protein 19. SppIP; activation peptide20. pSIP411 DNA 21. pSIP411 protein 22. PCR primer 23. PCR primer 24.pVAX1 DNA 25. pCTR DNA insert 26. pCXCL12 DNA insert

1. A plasmid which is capable of expressing a protein in lactic acidbacteria, wherein the said protein is selected from the group consistingof CXCL12, CXCL17 and Ym1.
 2. The plasmid of claim 1, wherein saidplasmid comprises a nucleotide sequence encoding a protein selectedfrom: (i) murine CXCL12-1α having an amino acid sequence as shown in SEQID NO: 3 or 2, or an amino acid sequence with at least 80% sequenceidentity thereto; (ii) human CXCL12-1α having an amino acid sequence asshown in SEQ ID NO: 6 or 5, or an amino acid sequence with at least 80%sequence identity thereto; (iii) murine CXCL17 having an amino acidsequence as shown in SEQ ID NO: 9 or 8, or an amino acid sequence withat least 80% sequence identity thereto; (iv) human CXCL17 having anamino acid sequence as shown in SEQ ID NO: 12 or 11, or an amino acidsequence with at least 80% sequence identity thereto; (v) murine Ym1having an amino acid sequence as shown in SEQ ID NO: 15 or 14, or anamino acid sequence with at least 80% sequence identity thereto; and(vi) human Ym1 as shown in SEQ ID NO: 18 or 17 or an amino acid sequencewith at least 80% sequence identity thereto.
 3. The plasmid according toclaim 1, wherein the plasmid comprises one or more regulatory sequenceswhich permit expression in lactic acid bacteria, wherein the regulatorysequences are obtained or derived from lactic acid bacteria.
 4. Theplasmid according to claim 1, wherein expression of said protein isregulatable.
 5. The plasmid according to claim 1, wherein the plasmidcomprises one or more nucleotide sequences encoding one or more of saidproteins under the control of an inducible promoter.
 6. The plasmidaccording to claim 1, wherein the plasmid comprises an induciblepromoter and regulatory elements from the nisin regulon, the sakacin Aregulon or the sakacin P regulon of a lactic acid bacterium.
 7. Theplasmid according to claim 5, wherein the inducible promoter is thePorfX promoter from the sakacin P regulon.
 8. The plasmid according toclaim 1, which is derived from the plasmid designated pSIP411.
 9. Therecombinant plasmid according to claim 1, wherein the nucleotidesequence encoding the protein is codon-optimised for expression inlactic acid bacteria.
 10. The plasmid according to claim 1, whichcomprises one or more nucleotide sequences selected from the groupconsisting of: a nucleotide sequence comprising the sequence of SEQ IDNO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQID NO: 16, or a nucleotide sequence having at least 80% sequenceidentity to any aforesaid sequence.
 11. A lactic acid bacterial straintransformed with the plasmid according to claim
 1. 12. The bacterialstrain according to claim 11, which is a Lactobacillus strain.
 13. Thebacterial strain according to claim 12, which is a strain ofLactobacillus reuteri.
 14. A wound dressing comprising the bacterialstrain according to claim
 11. 15. A pharmaceutical compositioncomprising the bacterial strain according to claim
 11. 16. The plasmidof claim 1 or a bacterial strain transformed with the plasmid for use intherapy.
 17. The plasmid of claim 1 or a bacterial strain transformedwith the plasmid for use in wound healing in a human or animal subject.18. The plasmid of claim 1 or a bacterial strain transformed with theplasmid for use in cutaneous or mucosal wound healing in a human oranimal subject.
 19. A kit for healing wounds, said kit comprising: (i)lactic acid bacteria comprising the plasmid of claim 1, wherein saidplasmid comprises a nucleotide sequence encoding a said protein underthe control of an inducible promoter capable of expressing the proteinin lactic acid bacteria; and (ii) an inducer for the promoter.
 20. Thekit of claim 19, being a pharmaceutical product comprising; (i) lacticacid bacteria comprising the plasmid of claim 1, wherein said plasmidcomprises a nucleotide sequence encoding a said protein under thecontrol of an inducible promoter capable of expressing the protein inlactic acid bacteria; and (ii) an inducer for the promoter as a combinedpreparation for separate, sequential or simultaneous use in woundhealing.
 21. A medical device comprising the bacterial strain accordingto claim
 11. 22. The bacterial strain of claim 11 wherein the bacteriaare freeze-dried.
 23. A kit for healing wounds, said kit comprising: thebacterial strain of claim 22, wherein the plasmid comprises a nucleotidesequence encoding the protein under the control of an inducible promotercapable of expressing the protein in lactic acid bacteria; an inducerfor the promoter; and a liquid for resuspending the freeze-driedbacteria.
 24. The kit of claim 23, wherein the liquid comprises theinducer.
 25. The kit of claim 19, wherein the kit comprises a wounddressing comprising the bacteria.
 26. The kit of claim 19, wherein thekit further comprises a wound dressing.